Isoprenoids are a class of natural products with more than 55,000 members. All isoprenoids are constructed from two precursors, isopentenyl diphosphate and its isomer dimethylallyl diphosphate. Two ...of the most important discoveries in isoprenoid biosynthetic studies in recent years are the elucidation of a second isoprenoid biosynthetic pathway the methylerythritol phosphate (MEP) pathway and a modified mevalonic acid (MVA) pathway. In this review, we summarize mechanistic insights on the MEP pathway enzymes. Because many isoprenoids have important biological activities, the need to produce them in sufficient quantities for downstream research efforts or commercial application is apparent. Recent advances in both MVA and MEP pathway-based synthetic biology are also illustrated by reviewing the landmark work of artemisinic acid and taxadien-5α-ol production through microbial fermentations.
A bio-based economy has the potential to provide sustainable substitutes for petroleum-based products and new chemical building blocks for advanced materials. We previously engineered Saccharomyces ...cerevisiae for industrial production of the isoprenoid artemisinic acid for use in antimalarial treatments. Adapting these strains for biosynthesis of other isoprenoids such as β-farnesene (C
H
), a plant sesquiterpene with versatile industrial applications, is straightforward. However, S. cerevisiae uses a chemically inefficient pathway for isoprenoid biosynthesis, resulting in yield and productivity limitations incompatible with commodity-scale production. Here we use four non-native metabolic reactions to rewire central carbon metabolism in S. cerevisiae, enabling biosynthesis of cytosolic acetyl coenzyme A (acetyl-CoA, the two-carbon isoprenoid precursor) with a reduced ATP requirement, reduced loss of carbon to CO
-emitting reactions, and improved pathway redox balance. We show that strains with rewired central metabolism can devote an identical quantity of sugar to farnesene production as control strains, yet produce 25% more farnesene with that sugar while requiring 75% less oxygen. These changes lower feedstock costs and dramatically increase productivity in industrial fermentations which are by necessity oxygen-constrained. Despite altering key regulatory nodes, engineered strains grow robustly under taxing industrial conditions, maintaining stable yield for two weeks in broth that reaches >15% farnesene by volume. This illustrates that rewiring yeast central metabolism is a viable strategy for cost-effective, large-scale production of acetyl-CoA-derived molecules.
Various types of cells are routinely cryopreserved in modern regenerative and cell-based medicines. For instance, the oocyte is one of the most demanding cells to be cryopreserved in genetic ...engineering and human-assisted reproductive technology (ART). However, the usage of cryopreserved oocytes in ART clinics is still limited mainly because of the unstable survival rate. This is due to the fact that oocytes are more prone to be damaged by ice crystals in comparison to other cells, as oocytes are larger in size and surface area. Meanwhile, oocytes contain more water, and thus, ice crystals are easier to form inside the cells. Currently, to avoid injury by the formed ice crystals, cryopreservation (CP) of oocytes has to use large amounts of small molecules as cryoprotectants such as dimethyl sulfoxide (DMSO) and ethylene glycol (EG), which can permeate into the cell and prevent ice formation inside. However, these molecules are chemically and epigenetically toxic to cells. Therefore, great efforts have been focused on reducing the amount of DMSO and EG used for oocyte CP. In nature, the antifreeze (glyco)proteins (AFGPs) locate extracellularly with the ability to protect living organisms from freezing damage via controlling ice growth. Inspired by this, biocompatible and nontoxic L-proline oligomers (L-Pro n ), which have the same polyproline II helix structure as that of AFGPs, are first employed for the CP of oocytes. The experimental results reveal that L-Pro8 has a profound activity in inhibiting ice growth as that of AFGP8. Also, by the addition of 50 mM L-Pro8, the amount of DMSO and EG can be greatly reduced by ca. 1.8 M for oocyte CP; moreover, the survival rate of the cryopreserved oocytes is increased up to 99.11%, and the coefficient of variance of the survival rate is decreased from 7.47 to 2.15%. These results mean that almost all oocytes can survive after CP with our method; importantly, the mitochondrial function as a critical criterion for the quality of the frozen–thawed oocytes is also improved. It is proposed that with the addition of L-Pro8, the extracellular ice growth is slowed down, which prevents the direct injuries of cells by large ice crystals and the accompanying osmotic pressure increase. As such, this work is not only significant for meeting the ever-increasing demand by the ART clinics but also gives guidance for designing materials in controlling ice growth during CP of other cells and tissues.
A compact broadband combiner with a high power capacity and a low insertion loss, which is especially useful for solid-state power sources where multi-way power synthesis is needed, was designed and ...experimentally investigated. The combiner could combine the microwave signals of sixteen terminals into a single one and was based on a radial-line waveguide whose circumferential symmetry benefited the amplitude and phase consistency of the combiner. Simulation and experimental results showed that the prototype device, designed for S-band applications, exhibited a reflection coefficient S
< -20 dB in the range of 2.06-2.93 GHz, which corresponds to a relative bandwidth of approximately 34.6%. At 2.45 GHz, the phase imbalance was ±4.5° and the 16-way transmission coefficient was concentrated around -12.0~-12.3 dB. The insertion loss of the device at ambient and elevated temperatures was simulated and experimentally verified, which is of importance for the design of similar high-power microwave combiners. High-power tests proved that even without enforced wind or liquid cooling, the device can handle continuous power (CW) of at least 3.9 kW, which can be much enhanced by taking regular cooling measures. The combined features of the designed combiner suggest promising applications for power synthesis in high-power, solid-state RF sources.
Fundamentally different responses of a LiTaO 3 thin film detector are observed when it is subjected to short microwave pulses as the pulse intensity is altered over a wide range. We start from weak ...microwave pulses which lead to only trivial pyroelectric peak response. However, when the microwave pulses become intense, the normally expected pyroelectric signal seems to be suppressed and the sign of the voltage signal can even be completely changed. Analysis indicates that while the traditional pyroelectric model, which is a linear model and works fine for our data in the small regime, it does not work anymore in the large signal regime. Since the small-signal model is the key foundation of electromagnetic-wave sensors based on pyroelectric effects, such as pyroelectric infrared detecters, the observation in this work suggests that one should be cautious when using these devices in intense fields. In addition, the evolution of detector signal with respect to excitation strength suggests that the main polarisation process is changed in the large signal regime. This is of fundamental importance to the understanding on how crystalline solids interact with intense microwaves. Possible causes of the nonlinear behaviour is discussed.
Isoprenoids are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate ...pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages to isoprenoid synthesis via the DXP pathway, such as a higher theoretical yield, and it has long been a goal to transplant the pathway into yeast. In this work, we investigate and address barriers to DXP pathway functionality in S. cerevisiae using a combination of synthetic biology, biochemistry and metabolomics. We report, for the first time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway.
•A heterologous DXP pathway has been functionally expressed in the yeast cytosol.•Identification of two functional pathway enzymes, IspG and IspH, was key.•Further optimization enabled growth in the absence of the mevalonate pathway.
In a survey of microbial systems capable of generating unusual metabolite structural variability, Streptomyces venezuelae ATCC 15439 is notable in its ability to produce two distinct groups of ...macrolide antibiotics. Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide, whereas narbomycin and pikromycin are derived from the 14-membered ring macrolactone, narbonolide. This report describes the cloning and characterization of the biosynthetic gene cluster for these antibiotics. Central to the cluster is a polyketide synthase locus (pikA) that encodes a six-module system comprised of four multifunctional proteins, in addition to a type II thioesterase (TEII). Immediately downstream is a set of genes for desosamine biosynthesis (des) and macrolide ring hydroxylation. The study suggests that Pik TEII plays a role in forming a metabolic branch through which polyketides of different chain length are generated, and the glycosyl transferase (encoded by desVII) has the ability to catalyze glycosylation of both the 12- and 14-membered ring macrolactones. Moreover, the pikC-encoded P450 hydroxylase provides yet another layer of structural variability by introducing regiochemical diversity into the macrolide ring systems. The data support the notion that the architecture of the pik gene cluster as well as the unusual substrate specificity of particular enzymes contributes to its ability to generate four macrolide antibiotics.
In vitro catalytic activity of DesVII, the glycosyltransferase involved in the biosynthesis of methymycin, neomethymycin, narbomycin, and pikromycin in Streptomyces venezuelae, is described. This is ...the first report of demonstrated in vitro activity of a glycosyltransferase involved in the biosynthesis of macrolide antibiotics. DesVII is unique among glycosyltransferases in that it requires an additional protein component, DesVIII, as well as basic pH for its full activity.
To understand the hang-up characteristics of caved ore and rock in caving mining under different macro factors, and to provide better guidance for drawpoint layout design and hang-up frequency ...prediction, numerical tests were conducted on an isolated draw model to investigate the influence of particle size, friction property, and overburden stress based on a rolling resistance model. A 3D phase diagram of hang-ups was obtained. Furthermore, the structural and mechanical characteristics of the local packing system of caved ore and rock under different hang-up and flow states were analyzed using statistical mechanics. The findings indicate that: (1) The proportion of hang-up events which is influenced by several factors decreases as the ratio of drawpoint size to average particle size increases, but increases with higher particle friction coefficient and overburden stress. And the ratio of drawpoint size to average particle size has the most significant impact on proportion of hang-up events, followed by particle friction coefficient, while overburden stress has the least impact. (2) The ratio of drawpoint size to average particle size primarily affects hang-ups by influencing average force chain strength, the particle friction coefficient mainly affects hang-ups through its influence on collimation coefficient of force chain and void ratio, and the overburden stress primarily affects hang-ups through its influence on the average force chain strength and the number of force chains. The average force chain strength and number of force chains in the local packing system of caved ore and rock serve as crucial meso-mechanical parameters that affect hang-ups.
Highlights
A 3D phase diagram of hang-ups was obtained.
The particle size most affects hang-up frequency, followed by particle friction coefficient and finally overburden stress.
The strength and number of force chains serve as crucial meso-mechanical parameters that affect hang-ups.
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