Isoprene Synthesis Research Articles

Enhancing isoprene production by supplementing mevalonate pathway expressed in E. coli with immobilized enzymes.

Isoprene is an important component in rubber production, which can be produced using the E. coli mevalonic acid (MVA) pathway, and this method has the advantage of green environmental protection and sustainable. However, due to the excessive accumulation of intermediates, the growth of cells was inhibited and the enzyme activity decreased gradually, so it was difficult to increase the yield of isoprene. The immobilized enzyme has the characteristics of high stability and strong reusability, so in this study, the immobilized enzyme was added to the fermentation process of isoprene production by mevalonate metabolizing bacteria (PT-P), to explore the effect on isoprene synthesis. Under the optimum conditions, compared with PT-P fermentation alone, the enzyme catalyzes the conversion of MVA with an efficiency of up to 50.86%, and the yield of isoprene increased by about 30%, reaching 234.47mg/L.

Limitations of Plant Stress Tolerance upon Heat and CO2 Exposure in Black Poplar: Assessment of Photosynthetic Traits and Stress Volatile Emissions.

Volatile organic compounds (VOCs) emitted by plants may help in understanding the status of a plant's physiology and its coping with mild to severe stress. Future climatic projections reveal that shifts in temperature and CO2 availability will occur, and plants may incur the uncoupling of carbon assimilation and synthesis of key molecules. This study explores the patterns of emissions of key VOCs (isoprene, methanol, acetaldehyde, and acetic acid) emitted by poplar leaves (more than 350) under a combined gradient of temperature (12-42 °C) and air CO2 concentration (400-1500 ppm), along with measurements of photosynthetic rates and stomatal conductance. Isoprene emission exhibited a rise with temperature and CO2 availability, peaking at 39 °C, the temperature at which methanol emission started to peak, illustrating the limit of stress tolerance to severe damage. Isoprene emission was uncoupled from the photosynthesis rate, indicating a shift from the carbon source for isoprene synthesis, while assimilation was decreased. Methanol and acetaldehyde emissions were correlated with stomatal conductance and peaked at 25 °C and 1200 ppm CO2. Acetic acid emissions lacked a clear correlation with stomatal conductance and the emission pattern of its precursor acetaldehyde. This study offers crucial insights into the limitations of photosynthetic carbon and stress tolerance.

The Active-Site [4Fe-4S] Cluster in the Isoprenoid Biosynthesis Enzyme IspH Adopts Unexpected Redox States during Ligand Binding and Catalysis.

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate reductase, or IspH (formerly known as LytB), catalyzes the terminal step of the bacterial methylerythritol phosphate (MEP) pathway for isoprene synthesis. This step converts (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into one of two possible isomeric products, either isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). This reaction involves the removal of the C4 hydroxyl group of HMBPP and addition of two electrons. IspH contains a [4Fe-4S] cluster in its active site, and multiple cluster-based paramagnetic species of uncertain redox and ligation states can be detected after incubation with reductant, addition of a ligand, or during catalysis. To characterize the clusters in these species, 57Fe-labeled samples of IspH were prepared and studied by electron paramagnetic resonance (EPR), 57Fe electron-nuclear double resonance (ENDOR), and Mössbauer spectroscopies. Notably, this ENDOR study provides a rarely reported, complete determination of the 57Fe hyperfine tensors for all four Fe ions in a [4Fe-4S] cluster. The resting state of the enzyme (Ox) has a diamagnetic [4Fe-4S]2+ cluster. Reduction generates [4Fe-4S]+ (Red) with both S = 1/2 and S = 3/2 spin ground states. When the reduced enzyme is incubated with substrate, a transient paramagnetic reaction intermediate is detected (Int) which is thought to contain a cluster-bound substrate-derived species. The EPR properties of Int are indicative of a 3+ iron-sulfur cluster oxidation state, and the Mössbauer spectra presented here confirm this. Incubation of reduced enzyme with the product IPP induced yet another paramagnetic [4Fe-4S]+ species (Red+P) with S = 1/2. However, the g-tensor of this state is commonly associated with a 3+ oxidation state, while Mössbauer parameters show features typical for 2+ clusters. Implications of these complicated results are discussed.

Inhibition of DXR in the MEP pathway with lipophilic N-alkoxyaryl FR900098 analogs.

In Mycobacterium tuberculosis (Mtb) and Plasmodium falciparum (Pf), the methylerythritol phosphate (MEP) pathway is responsible for isoprene synthesis. This pathway and its products are vital to bacterial/parasitic metabolism and survival, and represent an attractive set of drug targets due to their essentiality in these pathogens but absence in humans. The second step in the MEP pathway is the conversion of 1-deoxy-d-xylulose-5-phosphate (DXP) to MEP and is catalyzed by 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR). Natural products fosmidomycin and FR900098 inhibit DXR, but are too polar to reach the desired target inside some cells, such as Mtb. Synthesized FR900098 analogs with lipophilic substitution in the position α to the phosphorous atom showed promise, resulting in increased activity against Mtb and Pf. Here, an α substitution, consisting of a 3,4-dichlorophenyl substituent, in combination with various O-linked alkylaryl substituents on the hydroxamate moiety is utilized in the synthesis of a novel series of FR900098 analogs. The purpose of the O-linked alkylaryl substituents is to further enhance DXR inhibition by extending the structure into the adjacent NADPH binding pocket, blocking the binding of both DXP and NADPH. Of the initial O-linked alkylaryl substituted analogs, compound 6e showed most potent activity against Pf parasites at 3.60 μM. Additional compounds varying the phenyl ring of 6e were synthesized. The most potent phosphonic acids, 6l and 6n, display nM activity against PfDXR and low μM activity against Pf parasites. Prodrugs of these compounds were less effective against Pf parasites but showed modest activity against Mtb cells. Data from this series of compounds suggests that this combination of substituents can be advantageous in designing a new generation of antimicrobials.

Omics integration for in-depth understanding of the low-carbon co-culture platform system of Chlorella vulgaris-Escherichia coli

Many fuels and chemicals can be commercially produced by microbial processes to substitute the petrochemical processes. However, the microbial processes suffer from the instability, low efficiency or discontinuous cultures. The continuous culture can greatly increase the productivity and reduce costs for the microbial production. The challenges for the application of continuous culture in industry are high contamination risk and strain degeneration. The co-culture system of photoautotrophs and heterotrophs is probably able to solve bottlenecks of microbial culture. The micro-ecological balance can be achieved for the continuous microbial culture by the co-culture of photoautotrophs and heterotrophs. Here, a system for the co-cultivation of Chlorella vulgaris and Escherichia coli was established for the biosynthesis of isoprene. Compared with axenic culture, the isoprene production in the co-culture process was improved 10-fold to 0.6 g/L and the fermentation was prolonged from 100 h to 350 h. C. vulgaris promoted the isoprene synthesis and E. coli growth, while E. coli restricted C. vulgaris growth. The interactions between E. coli and C. vulgaris were closely associated with oxidative pressure from photomixotrophic metabolism. The consumption of exogenous glucose resulted in excess photomixotrophic electrons and subsequently resultant toxic reactive oxygen species (ROS). The oxidative pressure was reflected by the high activity of intracellular antioxidative (CysK, CysE, SerA, AhpC, AhpF) and repair (YtfE, NfuA, YebG) systems. C. vulgaris might protect E. coli against the oxidative pressure and improve the growth of E. coli through the inter-species cross-feeding. The biosynthesis of cysteine was greatly up-regulated in C. vulgaris to reduce ROS, and the cysteine necessary for antioxidation in E. coli might be provided by C. vulgaris. This study on the co-culture of C. vulgaris and E. coli is significant for revealing the common interactions between photoautotrophs and heterotrophs for the continuous culture.

Sensitivity of isoprene emission rate to ozone in greening trees is concurrently determined by isoprene synthesis capacity and stomatal conductance

The sensitivity of isoprene emission rate (ISOrate) to ozone (O3) in plant suggests potentially large changes in future isoprene emissions, which will have important consequences for atmospheric chemistry. However, the interspecific variation of ISOrate sensitivity to O3 and its key drivers remain largely unknown. In this study, four urban greening tree species were exposed to two O3 treatments (charcoal-filtered air, CF; and non-filtered ambient air plus 60 ppb extra O3, EO3) in open-top chambers for one growing season. We aimed to compare the interspecific variation in O3 inhibitory effect on ISOrate and explore its physiological mechanism. EO3 decreased the ISOrate by on average 42.5 % across species. According to absolute effect size ranking, the highest ISOrate sensitivity to EO3 was observed in Salix matsudana, followed by Sophora japonica and hybrid poplar clone ‘546’, while Quercus mongolica ISOrate was the least sensitive. Leaf anatomical structures differed in tree species but did not respond to EO3. Furthermore, the ISOrate sensitivity to O3 was driven by the concurrent effects of O3 on ISO synthesis ability (i.e., dimethylallyl diphosphate and isoprene synthase contents) and stomatal conductance. Overall, the mechanistic understanding grained from this study may promote the integrity of O3 effect into process-based ISO emission models.

Characterizing isoprene production in cyanobacteria – Insights into the effects of light, temperature, and isoprene on Synechocystis sp. PCC 6803

Engineering cyanobacteria for the production of isoprene and other terpenoids has gained increasing attention in the field of biotechnology. Several studies have addressed optimization of isoprene synthesis in cyanobacteria via enzyme and pathway engineering. However, only little attention has been paid to the optimization of cultivation conditions. In this study, an isoprene-producing strain of Synechocystis sp. PCC 6803 and two control strains were grown under a variety of cultivation conditions. Isoprene production, as quantified by modified membrane inlet mass spectrometer (MIMS) and interpreted using Flux Balance Analysis (FBA), increased under violet light and at elevated temperature. Increase of thermotolerance in the isoprene producer was attributed to the physical presence of isoprene, similar to plants. The results demonstrate a beneficial effect of isoprene on cell survival at higher temperatures. This increased thermotolerance opens new possibilities for sustainable bio-production of isoprene and other products.

Role of regulatory pathways and multi-omics approaches for carbon capture and mitigation in cyanobacteria

Cyanobacteria are known for their metabolic potential and carbon capture and sequestration capabilities. These cyanobacteria are not only an effective source for carbon minimization and resource mobilization into value-added products for biotechnological gains. The present review focuses on the detailed description of carbon capture mechanisms exerted by the various cyanobacterial strains, the role of important regulatory pathways, and their subsequent genes responsible for such mechanisms. Moreover, this review will also describe effectual mechanisms of central carbon metabolism like isoprene synthesis, ethylene production, MEP pathway, and the role of Glyoxylate shunt in the carbon sequestration mechanisms. This review also describes some interesting facets of using carbon assimilation mechanisms for valuable bio-products. The role of regulatory pathways and multi-omics approaches in cyanobacteria will not only be crucial towards improving carbon utilization but also will give new insights into utilizing cyanobacterial bioresource for carbon neutrality.

Beyond the central dogma with digital biochemistry

Isoprene as a bio-molecule may the simplest non-protein enzyme that spoiling pathogens and this Isoprene synthesis mostly by all the phytoplankton and specific terrestrial plants are mere environmentally supported resists diseases. Along with Isoprene all essential long chain Fatty acids and oils too are environmentally controlled bio-molecules that immensely protecting the diseases-prone of all the proteins, hence we say that the stated this concept as beyond the central dogma and unavoidable in long years to come to save the esteem mankind. According to the classical and principle studies natural migrations with temperature may help to achieve a longer or shorter life-spans according to temperature related bonding of molecular biochemistry and again isoprene, fats and oils environmentally protect them. Prevailing of pathogenic found linearity in relations to temperature and nitrogen sources or protein sources in respective hosts. In cool temperature peptide-bonds remains relatively cohesive in freezing and cool temperature, hence individual life-span can become more in cooler climate which prevail in higher altitudes. There are principles, lowering temperature, or else lowering protein-rich diets may make individual life-span extendable and worthy. A higher protein may invite many diseases in tropical climate and hence we advise to follow specific Amino-acids’ therapy to the mankind. We observe that all herbivore species remain diseases-less compare to carnivore species and also diseases in cold waters fisheries far lower than warm waters. In animal kingdom most fishes migrate towards the low temperature to gain a longer life-spans, whereas shellfishes, crabs or insects may migrate to higher temperature to softening their peptides and new births.

Clean synthesis of isoprene with six E. coli engineering strains

Isoprene is one of the most important platform chemicals containing conjugated double bonds and utilized as the precursor material of varieties of chemical raw materials. Currently, isoprene is mostly produced by petroleum smelting, which produces CO2 and some pollution. In that case, the clean production method of isoprene is still the focus of current research. Isoprene synthase (IspS) catalyzes isoprene formation from a metabolic intermediate DMAPP. The expression level and enzyme activity of IspS from different strains affect the amount of isoprene production. In this study, six IspS protein sequences were obtained by searching literatures and NCBI database based on the phylogenetic analysis. We analysed the conservation of their catalytic structures and constructed pBAD expression frameworks for them. The isoprene synthesis systems in E. coli were built and worked in the best induction condition predicted in our study. We used toluene to collect the isoprene produced in the system detected and compared the productivity of each strain. Our results show that the production in cells were 1.55-5.51 μg/L/h.

Analysis of the initial response pattern of <italic>Chlamydomonas reinhardtii</italic> to methyl jasmonate (MeJA) treatment

As a chemical elicitor of plants, methyl jasmonate (MeJA) is a regulator of many secondary metabolisms in plants, which participates in the regulation of isoprenoid metabolism and has an important impact on the synthesis of isoprenoids in <italic>Chlamydomonas reinhardtii</italic> (<italic>C. reinhardtii</italic>). In order to explore how <italic>C. reinhardtii</italic> responds rapidly to MeJA treatment, the physiological parameters such as growth rate and photosynthetic efficiency of <italic>C. reinhardtii</italic> under MeJA treatment were measured. The changes in the expression levels of key genes in the 2-C-methnatureyl-D-rythritol-4-phosphate (MEP) pathway and in the carotenoid and sterol synthesis pathway within 24 hours of MeJA treatment were focused. The results showed that MeJA treatment dramatically reduced biomass accumulation and photosynthetic activity of <italic>C. reinhardtii</italic>, prominently activated the expression of key genes in MEP pathway and sterol synthesis, inhibited key genes of carotenoid synthesis pathway. The results at the transcriptional and physiological levels suggested that the influence of MeJA on <italic>C. reinhardtii</italic> was rapid and dose-dependent. This study provides a theoretical reference for exploring the mechanism of isoprene synthesis and metabolism in microalgae.

Non-enzymatic formation of isoprene and 2-methyl-3-buten-2-ol (2-MBO) by manganese

It has been suggested that isoprene synthesis by isoprene synthase (IspS) proceeds via a substrate-assisted mechanism. The authors observed a non-enzymatic isoprene formation by Mn2+, which represents the basis of IspS enzyme reaction. Because IspS and many other terpene synthases require Mn2+ metal ions as cofactor, this study characterized the formation reaction for the first time. Metal ions including Mn2+ non-enzymatically produced both isoprene and 2-methyl-3-buten-2-ol (2-MBO) from dimethylallyl pyrophosphate (DMADP). Isoprene formation was most enhanced by Fe2+ and, to a lesser extent, by Mn2+ or Cu2+. Ni2+, Co2+, Mg2+, and Ba2+ exhibited a low activity to generate both isoprene and 2-MBO. The proportion of isoprene and 2-MBO varied with the Mn2+ concentration: isoprene predominated over 2-MBO at a higher Mn2+ concentration. Similarly, isoprene formation by Mn2+ increased exponentially as temperature increased with predominance of isoprene over 2-MBO at higher temperature. Both isoprene and 2-MBO formation was enhanced by acidic and neutral pH compared to alkaline conditions. Molecular dynamic simulation of DMADP suggested that the formation reaction is initiated by deprotonation of hydrogen on allyl terminal carbon by phosphate oxygen and generates carbocation and allyl anion intermediates. This is followed by quenching to produce isoprene or by hydroxyl addition to form 2-MBO. Thus, this study provided an insight into reaction mechanism of isoprene and 2-MBO biosynthesis and highlighted some parts of isoprene emission from terrestrial plants, which could be formed by non-enzymatic mechanism.

Genetic evidence for the requirements of antroquinonol biosynthesis by Antrodia camphorata during liquid-state fermentation.

The solid-state fermentation of Antrodia camphorata could produce a variety of ubiquinone compounds, such as antroquinonol (AQ). However, AQ is hardly synthesized during liquid-state fermentation (LSF). To investigates the mechanism of AQ synthesis, three precursors (ubiquinone 0 UQ0, farnesol and farnesyl diphosphate FPP) were added in LSF. The results showed that UQ0 successfully induced AQ production; however, farnesol and FPP could not induce AQ synthesis. The precursor that restricts the synthesis of AQ is the quinone ring, not the isoprene side chain. Then, the Agrobacterium-mediated transformation system of A. camphorata was established and the genes for quinone ring modification (coq2-6) and isoprene synthesis (HMGR, fps) were overexpressed. The results showed that overexpression of genes for isoprene side chain synthesis could not increase the yield of AQ, but overexpression of coq2 and coq5 could significantly increase AQ production. This is consistent with the results of the experiment of precursors. It indicated that the A. camphorata lack the ability to modify the quinone ring of AQ during LSF. Of the modification steps, prenylation of UQ0 is the key step of AQ biosynthesis. The result will help us to understand the genetic evidence for the requirements of AQ biosynthesis in A. camphorata.

The digital theories of isoprene nano-particle and other related in curing, preventing diseases caused by unicellular pathogens even in fisheries and allieds sciences during and after the Covid era

The digital rules of Isoprene can be proportionately prevention of diseases with temperature, atmospheric concentrations and altitude, since its holiness among the many natural bio-molecules. Its rule in preventing and curing diseases functionality are as proportionate to the temperature, air density, altitude etc. Isoprene (C5H8) is the bio-molecule helps in synthesising fatty acids, vitamins, steroids, cholesterol, secondary hormone, and sex hormones and many other micro or macro bio-molecules to the biological systems. Both plants and animal can synthesise Isoprene, often a few plants are medicinal because of Isoprene synthesis as well. Predominantly marine and terrestrial phytoplankton synthesize Isoprene in a large extents. Other terrestrial plants like Mosses, Pine tree, Grasses, Bamboo tree, Eucalyptus, Mango, Citrus spp. etc. can adequately synthesise Isoprene and makes the environment hygienic or pathogen free. As evidences found that Isoprene bio-molecule a nano-particle a smallest unit is below 3nm, can prevent and cure any unicellular pathogen by molecular diffusion. Isoprene firstly a water insoluble or hydrophobic molecule that repulse a hydrophilic pathogen. Secondly Isoprene having double bonds get saturated or hydrated with acid or even nucleic acids of unicellular pathogens, also assumed evidence may be found that isoprene can react with pathogenic nucleic acids or genetic material by forming Isoprene phosphate. Hence Isoprene can breakdown genetic materials of pathogens of unicellular simply by molecular diffusion within the pathogenic cells. In normal instances Isoprene can not penetrate in multi-cellular organisms. Often the oceans, or the Nilgiri hills in India, are so colourful and diseases less because of persisting Isoprene. In fisheries science we find that Isoprene is water insoluble with molecular density 0.68 gm/cc can be floated in water below 30 degree hence often fish species Catla catla or any top feeder can further remain diseases-less. In cold water Fisheries below 35o Celsius which is the vapour point of Isoprene and hence fisheries is safer, where as climates of tropics or global warming can lead isoprene less in waters but happy and hygiene terrestrial environment with Isoprene bio-molecule. May be true that Isoprene can destroy any mutants of viruses particles in a similar methodical phenomenon.

A Study of the Catalytic Properties of Synthetic Aluminosilicates of Different Morphologies in Cracking Reactions of the Byproducts of Isoprene Synthesis

The catalytic activity of synthetic samples of aluminosilicates of various morphologies and chemical compositions in a cascade of reactions that are byproducts (BPs) in the decomposition process of 4,4‑dimethyl-1,3-dioxane is studied. Aluminosilicates with montmorillonite structures (Na0.2Al1.8Mg0.2Si4O10(OH)2⋅nH2O, Mg3Si4O10(OH)2⋅nH2O), and kaolinite (Al2Si2O5(OH)4) with the spherical and platy morphologies of particles, as well as their porous-textural characteristics and surface properties, are studied. It is shown that the specific surface area of the studied samples, depending on the composition, varies from 11 to 470 m2/g, and the content of aluminum oxide in the samples ranged from 0 to 24 wt %. It is found that layered silicates with a montmorillonite structure contribute to reducing the gasification of organic raw materials and increasing the yield of isoprene. Kaolinite with a spherical morphology of particles increases the yield of formaldehyde, and with a platy morphology of particles, it increases the degree of decomposition of heteroatomic and cyclic compounds.

Study on heteropolyacid-based catalysts with high activity and reusability for isoprene synthesis from formaldehyde and isobutene

SiW/S-1 catalysts exhibited excellent catalytic activity and reusability due to the highly dispersed SiW and MFI structure of S-1.

Isoprene Emission in Darkness by a Facultative Heterotrophic Green Alga.

Isoprene is a highly reactive biogenic volatile hydrocarbon that strongly influences atmospheric oxidation chemistry and secondary organic aerosol budget. Many phytoplanktons emit isoprene like terrestrial pants. Planktonic isoprene emission is stimulated by light and heat and is seemingly dependent on photosynthesis, as in higher plants. However, prominent isoprene-emitting phytoplanktons are known to survive also as mixotrophs and heterotrophs. Chlorella vulgaris strain G-120, a unicellular green alga capable of both photoautotrophic and heterotrophic growth, was examined for isoprene emission using GC-MS and real-time PTR-MS in light (+CO2) and in darkness (+glucose). Chlorella emitted isoprene at the same rate both as a photoautotroph under light, and as an exclusive heterotroph while feeding on exogenous glucose in complete darkness. By implication, isoprene synthesis in eukaryotic phytoplankton can be fully supported by glycolytic pathways in absence of photosynthesis, which is not the case in higher plants. Isoprene emission by chlorophyll-depleted mixotrophs and heterotrophs in darkness serves unknown functions and may contribute to anomalies in oceanic isoprene estimates.

Isoprene Synthesis from Formaldehyde and Isobutylene in the Presence of Aluminum- and Niobium-Containing BEA Catalysts

A single-stage gas-phase synthesis of isoprene from formaldehyde and isobutylene in the presence of Al–BEA and Nb–BEA zeolite catalysts containing different amounts of aluminum and niobium has been studied. The physicochemical properties of the catalysts have been studied by low-temperature nitrogen adsorption, infrared spectroscopy of adsorbed CO, X-ray fluorescence analysis, and ammonia TPD methods. Catalytic tests have shown that, at similar formaldehyde conversion values, the isoprene selectivity is higher in the presence of the Al-containing BEA catalysts. It has been found that Al–BEA with Si/Al = 12 exhibits the highest activity in isoprene synthesis.

Depositing Different Carbon Species on MoP to Enhance Its Activity for Isoprene Production in Different Ways

To study the effects of aromatic and olefinic carbon on isoprene synthesis, different carbon sources were used to prepare a variety of carbon deposited MoP catalysts for the condensation of isobute...

Chemical Structure of Catalysts for Processing of High-Boiling By-Products of Isoprene Synthesis

Phase composition of catalysts for the processing of high-boiling by-products of isoprene production from isobutylene and formaldehyde was determined. A comparison of the chemical structure of the most effective K-97 and K-15 catalysts made it possible to find the role of boron oxide in the decrease in the coke formation.