Isoprene Production Research Articles

Isoprene nitrates drive new particle formation in Amazon’s upper troposphere

New particle formation (NPF) in the tropical upper troposphere is a globally important source of atmospheric aerosols1, 2, 3–4. It is known to occur over the Amazon basin, but the nucleation mechanism and chemical precursors have yet to be identified2. Here we present comprehensive in situ aircraft measurements showing that extremely low-volatile oxidation products of isoprene, particularly certain organonitrates, drive NPF in the Amazonian upper troposphere. The organonitrates originate from OH-initiated oxidation of isoprene from forest emissions in the presence of nitrogen oxides from lightning. Nucleation bursts start about 2 h after sunrise in the outflow of nocturnal deep convection, producing high aerosol concentrations of more than 50,000 particles cm−3. We report measurements of characteristic diurnal cycles of precursor gases and particles. Our observations show that the interplay between biogenic isoprene, deep tropical convection with associated lightning, oxidation photochemistry and the low ambient temperature uniquely promotes NPF. The particles grow over time, undergo long-range transport and descend through subsidence to the lower troposphere, in which they can serve as cloud condensation nuclei (CCN) that influence the Earth’s hydrological cycle, radiation budget and climate1,4, 5, 6, 7–8.

Unveiling the organic chemical composition and sources of organic carbon in PM2.5 at an urban site in Greater Cairo (Egypt): A comprehensive analysis of primary and secondary compounds

This work presents an exhaustive chemical characterization of the organic fraction of fine particulate matter (PM2.5) collected at an urban site in the Greater Cairo Area, Egypt, one of the most polluted megacities in the world. An intensive 2-month sampling campaign was conducted at an urban site in Giza (Dokki), from November 26, 2019, to January 28, 2020. Daily (24-h integrated) PM2.5 filter samples were then analyzed for their carbonaceous (OC, EC) and organic fractions including primary (n-alkanes, phthalates, fatty acids, polycyclic aromatic hydrocarbons, hopanes, sugars, and sugar alcohols) and secondary (isoprene and β-caryophyllene oxidation products, and dicarboxylic acids) compounds. Average organic (OC) and elemental carbon (EC) concentrations were 17.8 ± 6.6 μg/m3 and 4.4 ± 1.5 μg/m3, respectively. Biomass burning was confirmed by high daily concentration levels of levoglucosan, mannosan, and galactosan (sum equals to 288 ng/m3). Road traffic was also highlighted by the relative abundance of tetracosane and a carbon preference index close to unity as well as by the concentration ratios of PAHs and hopanes. Moreover, phthalates were identified for the first time in Cairo with high concentrations (654 ng/m3) that might be attributable to open waste burning activities. Fatty acids and sugars were also investigated and assigned to cooking activities and primary biogenic sources, respectively. The average concentration of isoprene and β-caryophyllene oxidation products were 0.89 ± 0.83 ng/m3, and 0.01 ± 0.02 ng/m3, respectively. These low values are expected since no pine trees or even forests exist in Egypt. The macrotracer approach was employed alongside Monte Carlo simulation to identify sources of primary OC and evaluate the uncertainties associated with source attribution and OC reconstruction. The findings revealed a strong contribution from cooking (31% of observed OC) and biomass burning (18%), with median reconstructed OC levels showing significant uncertainty (64%) as expected.

Novel Method to Quantify Trace Amounts of Isoprene and Monoterpene Secondary Organic Aerosol-Markers in Antarctic Ice.

Biogenic volatile organic compounds (BVOCs) contribute to the formation of secondary organic aerosol (SOA) through atmospheric oxidation. Previously detected SOA-markers in northern hemisphere ice cores from Alaska, Greenland, Russia, and Switzerland indicate the transportation of isoprene and monoterpene oxidation products from their forestry sources to these glacial regions. Antarctica is geographically further removed from the BVOC's source, indicating significantly lower SOA-marker concentrations are likely in southern hemisphere ice cores. The aim of this study was to develop a sensitive mass-spectrometric method to detect and quantify low-abundance SOA-markers of isoprene and monoterpenes in ice core samples. Employment of a triple quadrupole HPLC-MS method enabled limit of detections in the range of 0.4-10 ppt for nine terrestrial SOA-markers and a marker of biomass burning, levoglucosan. Quantification was conducted in the multiple reaction monitoring mode with two specific transitions monitored for each target compound. Application of the developed method onto a section of a Jurassic ice core from Antarctica revealed the presence of seven of the target compounds: 2-methylerythritol, 2-methylglyceric acid, cis-pinonic acid, 3-methyl-1,2,3-butanetricarboxylic acid, pinolic acid, cis-norpinonic acid, and pinic acid. Repeatability ranged between 2.2% and 6.2%. This is the first time that such SOA-markers have been discovered and quantified in Antarctic ice.

Highly Efficient and Selective Synthesis of Renewable Isoprene Over Mo-Fe-O and MgO/Mg(OH)2 Composite Catalysts.

Synthesis of renewable isoprene continues to attract significant interest, yet catalysts still need improvement for industrial applications. We report herein an efficient and novel Prins tandem approach for highly selective production of isoprene from bio-based methanol and isobutene over Mo-Fe-O and MgO/Mg(OH)2 acid-base bifunctional catalysts. Hydration decreases the number of strong basic sites O2- ions and increases OH groups over MgO, thus improving the isoprene selectivity. Basic OH on Mg(OH)2 promotes the Prins condensation of formaldehyde and isobutene, while acidic OH promotes dehydration of isoprenol to isoprene. Suitable contents and proportions of basic and acidic OH groups over Mg(OH)2 increase isoprene selectivity and methanol conversion to 90 and 92 % on the Mo-Fe-O+Mg(OH)2 composite catalysts, respectively. It exhibits good recycling stability, with isoprene selectivity remaining 90 % after five successive regenerations. DFT calculations reveal OH groups formed after hydration reduce the energy barrier of the H transfer step, thereby promoting the overall reaction.

Spatiotemporal variations of PM2.5 organic molecular markers in five central cities of the Yangtze River Delta, East China in autumn and winter: Implications for regional and local sources of organic aerosols

Information on the spatiotemporal variations in the composition and sources of organic aerosols (OA) is needed to identify regional influences and to establish effective control measures. Here, 23-h PM2.5 samples were collected in five central cities of the Yangtze River Delta in eastern China, including Nanjing, Suzhou, Wuxi, Changzhou, and Zhenjiang, every three days from 2020/09/01 to 2021/02/28. Each sample was analyzed for water-soluble inorganic ions, organic carbon (OC), elemental carbon (EC), and organic molecular markers (OMMs). Generally, the major components of PM2.5, including NH4+, SO42−, NO3−, OC, and EC, exhibited similar temporal patterns across the five cities. In all OMM groups, the concentrations of PAHs, oxygenated PAHs, and secondary products of isoprene showed strong correlations (r = 0.79 ± 0.050–0.93 ± 0.028) and low coefficient of divergence (COD = 0.22 ± 0.024–0.30 ± 0.033) between sampling sites, indicating a homogeneous spatial distribution of industrial emissions and biogenic secondary OA in autumn and winter. Other OMMs showed wider r (e.g., steranes and hopanes, 0.20–0.80) and COD (0.26–0.69) ranges for all site pairs, probably due to the influence of local emissions. Based on the source apportionment results using Positive matrix factorization, the biomass burning factor dominated the contribution to OC and EC in winter and showed strong correlations (r = 0.84 ± 0.063) between the sampling sites, indicating regional transport of emissions from biomass burning and fossil fuel combustion in the heating season. Traffic-related factors had the greatest spatial heterogeneity (r = 0.27 ± 0.19–0.51 ± 0.16) and contributed significantly to OC at their maximum levels.

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.

Isoprene emission dynamics in Chaetoceros curvisetus: Insights from transcriptome analysis under light/dark cycles

Isoprene from marine emissions significantly influences the atmospheric oxidative capacity and secondary pollutants in the marine boundary layer. While marine algae are recognized as primary contributors to marine emissions of isoprene, the long-term and dark-phase isoprene production from these organisms remains underexplored, potentially lead to inaccuracies in estimating marine isoprene emissions. In this study, we conducted a time series investigation of isoprene emission from Chaetoceros curvisetus with a growth cycle duration of 19 days and examined the influence of environmental factors. Our findings revealed that C. curvisetus emits isoprene during its whole growth cycle, with peak emissions (2.82 × 10−11 μmol cells−1 h−1) recorded in the stationary phase. Under nitrogen, light and temperature stress, significant release of isoprene is found. During both the light and dark phases, isoprene emission was observed with comparable intensities. Transcriptome analysis showed that 41 differentially expressed transcripts were involved in the synthesis of volatile organic compounds. Notably, downregulation of genes associated with pyruvate synthesis in the glycolysis pathway suggests its importance for dark isoprene release. The study also highlighted downregulation of isopentenyl phosphate kinase and Gene Ontology enrichment in organelles housing mevalonate (MVA) pathways, suggesting that exchange involving the transport of intermediate metabolites between MVA and methylerythritol phosphate (MEP) pathway may contribute to the released isoprene in dark phase. Furthermore, the synthesis of substrates and downstream products is related to the dark release of isoprene. In conclusion, our study illuminates the pivotal roles of substrate availability, interplay between MVA and MEP pathways, and glycolysis pathway in governing isoprene emissions under light/dark cycles.

Mutagenic atmospheres generated from the photooxidation of NOx with selected VOCs and a complex mixture: Apportionment of aromatic mutagenicity for reacted gasoline vapor

The interaction of sunlight with volatile organic compounds (VOCs) emitted from various sources results in mutagenic photooxidation products that contribute substantially to air pollution. Evaporation of gasoline is one such source of VOCs; however, no studies have evaluated the mutagenicity of the photooxidation products of gasoline vapors or of many of the non-aromatic constituent VOCs of gasoline. Here we determined the mutagenicity in Salmonella TA100 of atmospheres generated in a steady-state atmospheric simulation chamber by irradiating gasoline and individual non-aromatic VOCs in the presence of nitrogen oxides (NOX) in air. In addition to gasoline, we evaluated α-pinene; 2-pentene; ethanol; isobutanol; isoprene; and 2,2,4-trimethylpentane (isooctane). Cells were exposed at the air-agar interface to the atmospheres for 1, 2, 4, 8, or 16 h. Atmospheres generated in the dark were not mutagenic. However, under irradiation all atmospheres other than that of 2,2,4-trimethylpentane were mutagenic, with mutagenic potencies spanning 8.6-fold. The mutagenicity was due exclusively to direct-acting, late-generation photooxidation products. The non-aromatic VOCs studied here contributed little to the mutagenic potency of the photooxidation products of gasoline. However, the sum of the mutagenic potencies of these atmospheres plus those from the photooxidation of some aromatic VOCs in gasoline measured here and elsewhere (Riedel et al., 2018) accounted for 71% of the mutagenic potency of the photooxidation products of gasoline vapor. In photochemical mixtures with strong biogenic contributions, isoprene products may also contribute significantly to mutagenic potency. Strategies to reduce the emissions of gasoline and those VOCs whose photooxidation products are most mutagenic would reduce VOC-associated air pollution and improve public health.

Optimal seasonal schedule for the production of isoprene, a highly volatile biogenic VOC

The leaves of many trees emit volatile organic compounds (abbreviated as BVOCs), which protect them from various damages, such as herbivory, pathogens, and heat stress. For example, isoprene is highly volatile and is known to enhance the resistance to heat stress. In this study, we analyze the optimal seasonal schedule for producing isoprene in leaves to mitigate damage. We assume that photosynthetic rate, heat stress, and the stress-suppressing effect of isoprene may vary throughout the season. We seek the seasonal schedule of isoprene production that maximizes the total net photosynthesis using Pontryagin’s maximum principle. The isoprene production rate is determined by the changing balance between the cost and benefit of enhanced leaf protection over time. If heat stress peaks in midsummer, isoprene production can reach its highest levels during the summer. However, if a large portion of leaves is lost due to heat stress in a short period, the optimal schedule involves peaking isoprene production after the peak of heat stress. Both high photosynthetic rate and high isoprene volatility in midsummer make the peak of isoprene production in spring. These results can be clearly understood by distinguishing immediate impacts and the impacts of future expectations.

Enhancing bio-isoprene production in Escherichia coli through a combinatorial optimization approach

This study proposed a two-step process optimization for enhanced cumulative isoprene production. This involves establishing an isoprene biosynthetic pathway in Escherichia coli BL21 DE-3 via up-regulation of native deoxy xylulose 5-phosphate (DXP) synthase (EcDXS), isopentenyl pyrophosphate isomerase (EcIPPI/EcIDI) and introducing isoprene synthase (PmIspS) from kudzu (Pueraria montana), followed by the process conditions (incubation temperature, time, and inducer concentration) optimization using the Box-Behnken design (BBD) approach. BBD showed the maximum cumulative isoprene production (160.15 mg L-1), productivity (11.18 mg L-1 h-1), and yield (7.69 mg gdcw-1) at the optimized process conditions (incubation temperature 27.32 ºC, incubation time 14.25 h, and inducer concentration 0.453 mM). The transgenic E. coli isoprene production, -productivity, and -yield were 1.52-, 1.46-, and 1.24-fold higher than the unoptimized condition (incubation temperature 30 ºC, incubation time 16 h, and inducer concentration 0.10 mM). This work demonstrates that fine tuning of MEP pathway in addition with process conditions optimization is an efficient strategy for improving isoprene production from engineered E. coli.

Unravelling the origin of isoprene in the human body—a forty year Odyssey

In the breath research community’s search for volatile organic compounds that can act as non-invasive biomarkers for various diseases, hundreds of endogenous volatiles have been discovered. Whilst these systemic chemicals result from normal and abnormal metabolic activities or pathological disorders, to date very few are of any use for the development of clinical breath tests that could be used for disease diagnosis or to monitor therapeutic treatments. The reasons for this lack of application are manifold and complex, and these complications either limit or ultimately inhibit the analytical application of endogenous volatiles for use in the medical sciences. One such complication is a lack of knowledge on the biological origins of the endogenous volatiles. A major exception to this is isoprene. Since 1984, i.e. for 40 years, it has been generally accepted that the pathway to the production of human isoprene, and hence the origin of isoprene in exhaled breath, is through cholesterol biosynthesis via the mevalonate (MVA) pathway within the liver. However, various studies between 2001 and 2012 provide compelling evidence that human isoprene is produced in skeletal muscle tissue. A recent multi-omic investigation of genes and metabolites has revealed that this proposal is correct by showing that human isoprene predominantly results from muscular lipolytic cholesterol metabolism. Despite the overwhelming proof for a muscular pathway to isoprene production in the human body, breath research papers still reference the hepatic MVA pathway. The major aim of this perspective is to review the evidence that leads to a correct interpretation for the origins of human isoprene, so that the major pathway to human isoprene production is understood and appropriately disseminated. This is important, because an accurate attribution to the endogenous origins of isoprene is needed if exhaled isoprene levels are to be correctly interpreted and for assessing isoprene as a clinical biomarker.

Impact of persistently high sea surface temperatures on the rhizobiomes of Zostera marina in a Baltic Sea benthocosms.

Persistently high marine temperatures are escalating and threating marine biodiversity. The Baltic Sea, warming faster than other seas, is a good model to study the impact of increasing sea surface temperatures. Zostera marina, a key player in the Baltic ecosystem, faces susceptibility to disturbances, especially under chronic high temperatures. Despite the increasing number of studies on the impact of global warming on seagrasses, little attention has been paid to the role of the holobiont. Using an outdoor benthocosm to replicate near-natural conditions, this study explores the repercussions of persistent warming on the microbiome of Z. marina and its implications for holobiont function. Results show that both seasonal warming and chronic warming, impact Z. marina roots and sediment microbiome. Compared with roots, sediments demonstrate higher diversity and stability throughout the study, but temperature effects manifest earlier in both compartments, possibly linked to premature Z. marina die-offs under chronic warming. Shifts in microbial composition, such as an increase in organic matter-degrading and sulfur-related bacteria, accompany chronic warming. A higher ratio of sulfate-reducing bacteria compared to sulfide oxidizers was found in the warming treatment which may result in the collapse of the seagrasses, due to toxic levels of sulfide. Differentiating predicted pathways for warmest temperatures were related to sulfur and nitrogen cycles, suggest an increase of the microbial metabolism, and possible seagrass protection strategies through the production of isoprene. These structural and compositional variations in the associated microbiome offer early insights into the ecological status of seagrasses. Certain taxa/genes/pathways may serve as markers for specific stresses. Monitoring programs should integrate this aspect to identify early indicators of seagrass health. Understanding microbiome changes under stress is crucial for the use of potential probiotic taxa to mitigate climate change effects. Broader-scale examination of seagrass-microorganism interactions is needed to leverage knowledge on host-microbe interactions in seagrasses.

CAMx–UNIPAR simulation of secondary organic aerosol mass formed from multiphase reactions of hydrocarbons under the Central Valley urban atmospheres of California

Abstract. The UNIfied Partitioning-Aerosol phase Reaction (UNIPAR) model was integrated into the Comprehensive Air quality Model with extensions (CAMx) to process secondary organic aerosol (SOA) formation by capturing multiphase reactions of hydrocarbons (HCs) in regional scales. SOA growth was simulated using a wide range of anthropogenic HCs, including 10 aromatics and linear alkanes with different carbon lengths. The atmospheric processes of biogenic HCs (isoprene, terpenes, and sesquiterpene) were simulated for major oxidation paths (ozone, OH radicals, and nitrate radicals) to predict day and night SOA formation. The UNIPAR model streamlined the multiphase partitioning of the lumping species originating from semi-explicitly predicted gas products and their heterogeneous chemistry to form non-volatile oligomeric species in both organic aerosol and inorganic aqueous phase. The CAMx–UNIPAR model predicted SOA formation at four ground urban sites (San Jose, Sacramento, Fresno, and Bakersfield) in California, United States, during wintertime 2018. Overall, the simulated mass concentrations of the total organic matter, consisting of primary organic aerosol and SOA, showed a good agreement with the observations. The simulated SOA mass in the urban areas of California was predominated by alkane and terpene oxidation products. During the daytime, low-volatility products originating from the autoxidation of long-chain alkanes considerably contributed to the SOA mass. In contrast, a significant amount of nighttime SOA was produced by the reaction of terpene with ozone or nitrate radicals. The spatial distributions of anthropogenic SOA associated with aromatic and alkane HCs were noticeably affected by the southward wind direction, owing to the relatively long lifetime of their atmospheric oxidation, whereas those of biogenic SOA were nearly insensitive to wind direction. During wintertime 2018, the impact of inorganic aerosol hygroscopicity on the total SOA budget was not evident because of the small contribution of aromatic and isoprene products, which are hydrophilic and reactive in the inorganic aqueous phase. However, an increased isoprene SOA mass was predicted during the wet periods, although its contribution to the total SOA was little.

Decarbonizing gas emissions from petrochemical production using microalgae

Thermal and catalytic processes of purification of hydrocarbon raw materials, which are produced by oil refineries and petrochemical enterprises, make a significant contribution to the increase in greenhouse gas emissions. Exhaust gases of thermal and catalytic processes containing a mixture of inert gases and C1-C5 hydrocarbons are sent for flaring. Industrial enterprises, especially petrochemical ones, emit a large amount of other greenhouse gases besides CO2. This study was executed in order to study the absorption of blow-off gases obtained during petrochemical production by marine microalgae Isochrysis galbana and Chlorella microalgae. In each experiment conducted as part of this study, microalgae underwent two successive growth phases: the preparation phase and the cultivation phase. The studies were conducted at various temperatures and pressures. Exhaust and blow-off gases of the existing industrial production of isoprene were selected for laboratory experiments, so the composition of the gas changed significantly between tests. The microalgae showed the highest absorption capacity under the condition of 32 °C and high gas pressure. Microalgae Isochrysis galbana and Chlorella microalgae showed the ability to absorb gases C1–C5 with an efficiency of 75.0%. The obtained research results can be used in the complex cleaning of biological treatment facilities.

ISOLATION OF HIGH PURITY PIPERYLENE

Piperylene is a valuable raw material component for petrochemical synthesis, perfumery and cosmetic products, and is used as a solvent for petroleum products in the production of synthetic rubbers and latexes, and in the paint and varnish industry. Piperylene in the form of a fraction is produced in the production of isoprene by the two-stage dehydrogenation of isopentane, where it is a secondary product of the main synthesis. Isolation of commercial piperylene of high purity will allow the production of a value-added product and expand the sales market. Currently, restrictions on the production of commercial piperylene are associated with the high content of an impurity – cyclopentadiene. Previous studies by the author showed the feasibility of extracting cyclopentadiene at the stage of obtaining the catalyzate of the second stage of isoprene dehydrogenation. Simulation of the technological scheme in the application program showed that, together, the extraction of cyclopentadiene by a chemical method and a change in the technological operating mode makes it possible to isolate piperylene with a content of 99.45 % wt and CPD equal to 0.0046 % wt.

Isoprene Production and Its Driving Factors in the Northwest Pacific Ocean

AbstractMarine isoprene plays a crucial role in the formation of secondary organic aerosol within the remote marine boundary layer. Due to scarce field measurements of oceanic isoprene and limited laboratory‐based studies of isoprene production, assessing the importance of marine isoprene on atmospheric chemistry and climate is challenging. Calculating in‐field isoprene production rates is a crucial step to predict marine isoprene concentrations and the subsequent emissions to the atmosphere. The distribution, sources, and dominant environmental factors of isoprene were determined in the Northwest Pacific Ocean in 2019. The nutrient enrichment in the Kuroshio Oyashio Extension (KOE) surface seawater, driven by the upwelling and atmospheric deposition, promoted the growth of phytoplankton and elevated the isoprene concentration. This was confirmed by observed responses of isoprene to nutrients and aerosol dust additions in a ship‐based incubation experiment, where the isoprene concentrations increased by 70% (t = 4.417, p < 0.001) and 35% (t = 2.387, p < 0.05), respectively. Biogenic isoprene production rates in the deck incubation experiments were positively related to chlorophyll a, temperature, and solar radiation, with an average production of 7.33 ± 4.27 pmol L−1 day−1. Photochemical degradation of dissolved organic matter was likely an abiotic source of isoprene, contributing to approximately 14% of the total production. Driven by high isoprene production and extreme physical disturbance, the KOE showed very high emissions of isoprene of 46.0 ± 13.0 nmol m−2 day−1, which led to a significant influence on the oxidative capacity of the local atmosphere.

Engineered Methylococcus capsulatus Bath for efficient methane conversion to isoprene

Isoprene has numerous industrial applications, including rubber polymer and potential biofuel. Microbial methane-based isoprene production could be a cost-effective and environmentally benign process, owing to a reduced carbon footprint and economical utilization of methane. In this study, Methylococcus capsulatus Bath was engineered to produce isoprene from methane by introducing the exogenous mevalonate (MVA) pathway. Overexpression of MVA pathway enzymes and isoprene synthase from Populus trichocarpa under the control of a phenol-inducible promoter substantially improved isoprene production. M. capsulatus Bath was further engineered using a CRISPR-base editor to disrupt the expression of soluble methane monooxygenase (sMMO), which oxidizes isoprene to cause toxicity. Additionally, optimization of the metabolic flux in the MVA pathway and culture conditions increased isoprene production to 228.1 mg/L, the highest known titer for methanotroph-based isoprene production. The developed methanotroph could facilitate the efficient conversion of methane to isoprene, resulting in the sustainable production of value-added chemicals.

Optimization of IspSib stability through directed evolution to improve isoprene production.

Enzyme stability is often a limiting factor in the microbial production of high-value-added chemicals and commercial enzymes. A previous study by our research group revealed that the unstable isoprene synthase from Ipomoea batatas (IspSib) critically limits isoprene production in engineered Escherichia coli. Directed evolution was, therefore, performed in the present study to improve the thermostability of IspSib. First, a tripartite protein folding system designated as lac'-IspSib-'lac, which could couple the stability of IspSib to antibiotic ampicillin resistance, was successfully constructed for the high-throughput screening of variants. Directed evolution of IspSib was then performed through two rounds of random mutation and site-saturation mutation, which produced three variants with higher stability: IspSibN397V A476V, IspSibN397V A476T, and IspSibN397V A476C. The subsequent in vitro thermostability test confirmed the increased protein stability. The melting temperatures of the screened variants IspSibN397V A476V, IspSibN397V A476T, and IspSibN397V A476C were 45.1 ± 0.9°C, 46.1 ± 0.7°C, and 47.2 ± 0.3°C, respectively, each of which was higher than the melting temperature of wild-type IspSib (41.5 ± 0.4°C). The production of isoprene at the shake-flask fermentation level was increased by 1.94-folds, to 1,335 mg/L, when using IspSibN397V A476T. These findings provide insights into the optimization of the thermostability of terpene synthases, which are key enzymes for isoprenoid production in engineered microorganisms. In addition, the present study would serve as a successful example of improving enzyme stability without requiring detailed structural information or catalytic reaction mechanisms.IMPORTANCEThe poor thermostability of IspSib critically limits isoprene production in engineered Escherichia coli. A tripartite protein folding system designated as lac'-IspSib-'lac, which could couple the stability of IspSib to antibiotic ampicillin resistance, was successfully constructed for the first time. In order to improve the enzyme stability of IspSib, the directed evolution of IspSib was performed through error-PCR, and high-throughput screening was realized using the lac'-IspSib-'lac system. Three positive variants with increased thermostability were obtained. The thermostability test and the melting temperature analysis confirmed the increased stability of the enzyme. The production of isoprene was increased by 1.94-folds, to 1,335 mg/L, using IspSibN397V A476T. The directed evolution process reported here is also applicable to other terpene synthases key to isoprenoid production.

Origin of breath isoprene in humans is revealed via multi-omic investigations

Plants, animals and humans metabolically produce volatile isoprene (C5H8). Humans continuously exhale isoprene and exhaled concentrations differ under various physio-metabolic and pathophysiological conditions. Yet unknown metabolic origin hinders isoprene to reach clinical practice as a biomarker. Screening 2000 individuals from consecutive mass-spectrometric studies, we herein identify five healthy German adults without exhaled isoprene. Whole exome sequencing in these adults reveals only one shared homozygous (European prevalence: <1%) IDI2 stop-gain mutation, which causes losses of enzyme active site and Mg2+–cofactor binding sites. Consequently, the conversion of isopentenyl diphosphate to dimethylallyl diphosphate (DMAPP) as part of the cholesterol metabolism is prevented in these adults. Targeted sequencing depicts that the IDI2 rs1044261 variant (p.Trp144Stop) is heterozygous in isoprene deficient blood-relatives and absent in unrelated isoprene normal adults. Wild-type IDI1 and cholesterol metabolism related serological parameters are normal in all adults. IDI2 determines isoprene production as only DMAPP sources isoprene and unlike plants, humans lack isoprene synthase and its enzyme homologue. Human IDI2 is expressed only in skeletal-myocellular peroxisomes and instant spikes in isoprene exhalation during muscle activity underpins its origin from muscular lipolytic cholesterol metabolism. Our findings translate isoprene as a clinically interpretable breath biomarker towards potential applications in human medicine.

Oxidation characteristic and thermal runaway of isoprene

In this study, the oxidation characteristics of isoprene were investigated using a custom-designed mini closed pressure vessel test (MCPVT). The results show that isoprene is unstable and polymerization occurs under a nitrogen atmosphere. Under an oxygen atmosphere, the oxidation process of isoprene was divided into three stages: (1) isoprene reacts with oxygen to produce peroxide; (2) Peroxides produce free radicals through thermal decomposition; (3) Free radicals cause complex oxidation and thermal runaway reactions. The oxidation of isoprene conforms to the second-order reaction kinetics, and the activation energy was 86.88 kJ·mol−1. The thermal decomposition characteristics of the total oxidation product and purified peroxide mixture were determined by differential scanning calorimetry (DSC). The initial exothermic temperatures Ton were 371.17 K and 365.84 K, respectively. And the decomposition heat QDSC were 816.66 J·g−1 and 991.08 J·g−1, respectively. It indicates that high concentration of isoprene peroxide has a high risk of thermal runaway. The results of thermal runaway experiment showed that the temperature and pressure of isoprene oxidation were prone to rise rapidly, which indicates that the oxidation reaction was dangerous. The reaction products of isoprene were analyzed by gas chromatography-mass spectrometry (GC–MS). The main oxidation products were methyl vinyl ketone, methacrolein, 3-methylfuran, etc. The main thermal runaway products were dimethoxymethane, 2,3-pentanedione, naphthalene, etc. Based on the reaction products, the possible reaction pathway of isoprene was proposed.