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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., (© 2024. The Author(s).)

The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not well understood. Using metabolic control analysis (MCA), we examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), in multiple grey poplar ( Populus × canescens ) lines modified in their DXS activity. Single leaves were dynamically labeled with 13 CO 2 in an illuminated, climate-controlled gas exchange cuvette coupled to a proton transfer reaction mass spectrometer, and the carbon flux through the MEP pathway was calculated. Carbon was rapidly assimilated into MEP pathway intermediates and labeled both the isoprene released and the IDP+DMADP pool by up to 90%. DXS activity was increased by 25% in lines overexpressing the DXS gene and reduced by 50% in RNA interference lines, while the carbon flux in the MEP pathway was 25-35% greater in overexpressing lines and unchanged in RNA interference lines. Isoprene emission was also not altered in these different genetic backgrounds. By correlating absolute flux to DXS activity under different conditions of light and temperature, the flux control coefficient was found to be low. Among isoprenoid end products, isoprene itself was unchanged in DXS transgenic lines, but the levels of the chlorophylls and most carotenoids measured were 20-30% less in RNA interference lines than in overexpression lines. Our data thus demonstrate that DXS in the isoprene-emitting grey poplar plays only a minor part in controlling flux through the MEP pathway.

Many plants, especially trees, emit isoprene in a highly light- and temperature-dependent manner. The advantages for plants that emit, if any, have been difficult to determine. Direct effects on membranes have been disproven. New insights have been obtained by RNA sequencing, proteomic and metabolomic studies. We determined the responses of the phosphoproteome to exposure of Arabidopsis leaves to isoprene in the gas phase for either 1 or 5 h. Isoprene effects that were not apparent from RNA sequencing and other methods but were apparent in the phosphoproteome include effects on chloroplast movement proteins and membrane remodelling proteins. Several receptor kinases were found to have altered phosphorylation levels. To test whether potential isoprene receptors could be identified, we used molecular dynamics simulations to test for proteins that might have strong binding to isoprene and, therefore might act as receptors. Although many Arabidopsis proteins were found to have slightly higher binding affinities than a reference set of Homo sapiens proteins, no specific receptor kinase was found to have a very high binding affinity. The changes in chloroplast movement, photosynthesis capacity and so forth, found in this work, are consistent with isoprene responses being especially useful in the upper canopy of trees., (© 2023 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Isoprene is emitted by some plants and is the most abundant biogenic hydrocarbon entering the atmosphere. Multiple studies have elucidated protective roles of isoprene against several environmental stresses, including high temperature, excessive ozone, and herbivory attack. However, isoprene emission adversely affects atmospheric chemistry by contributing to ozone production and aerosol formation. Thus, understanding the regulation of isoprene emission in response to varying environmental conditions, for example, elevated CO 2 , is critical to comprehend how plants will respond to climate change. Isoprene emission decreases with increasing CO 2 concentration; however, the underlying mechanism of this response is currently unknown. We demonstrated that high-CO 2 -mediated suppression of isoprene emission is independent of photosynthesis and light intensity, but it is reduced with increasing temperature. Furthermore, we measured methylerythritol 4-phosphate (MEP) pathway metabolites in poplar leaves harvested at ambient and high CO 2 to identify why isoprene emission is reduced under high CO 2 . We found that hydroxymethylbutenyl diphosphate (HMBDP) was increased and dimethylallyl diphosphate (DMADP) decreased at high CO 2. This implies that high CO 2 impeded the conversion of HMBDP to DMADP, possibly through the inhibition of HMBDP reductase activity, resulting in reduced isoprene emission. We further demonstrated that although this phenomenon appears similar to abscisic acid (ABA)-dependent stomatal regulation, it is unrelated as ABA treatment did not alter the effect of elevated CO 2 on the suppression of isoprene emission. Thus, this study provides a comprehensive understanding of the regulation of the MEP pathway and isoprene emission in the face of increasing CO 2 .

Metabolomics studies are becoming increasingly common for understanding how plant metabolism responds to changes in environmental conditions, genetic manipulations and treatments. Despite the recent advances in metabolomics workflow, the sample preparation process still limits the high-throughput analysis in large-scale studies. Here, we present a highly flexible robotic system that integrates liquid handling, sonication, centrifugation, solvent evaporation and sample transfer processed in 96-well plates to automatize the metabolite extraction from leaf samples. We transferred an established manual extraction protocol performed to a robotic system, and with this, we show the optimization steps required to improve reproducibility and obtain comparable results in terms of extraction efficiency and accuracy. We then tested the robotic system to analyze the metabolomes of wild-type and four transgenic silver birch (Betula pendula Roth) lines under unstressed conditions. Birch trees were engineered to overexpress the poplar (Populus × canescens) isoprene synthase and to emit various amounts of isoprene. By fitting the different isoprene emission capacities of the transgenic trees with their leaf metabolomes, we observed an isoprene-dependent upregulation of some flavonoids and other secondary metabolites as well as carbohydrates, amino acid and lipid metabolites. By contrast, the disaccharide sucrose was found to be strongly negatively correlated to isoprene emission. The presented study illustrates the power of integrating robotics to increase the sample throughput, reduce human errors and labor time, and to ensure a fully controlled, monitored and standardized sample preparation procedure. Due to its modular and flexible structure, the robotic system can be easily adapted to other extraction protocols for the analysis of various tissues or plant species to achieve high-throughput metabolomics in plant research., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

In Ficus septica, the short-term control of isoprene production and, therefore, isoprene emission has been linked to the hormone balance between auxin (IAA) and jasmonic acid (JA). However, the relationship between long-term changes in isoprene emission and that of plant hormones remains unknown. This study tracked isoprene emissions from F. septica leaves, plant hormone concentrations and signalling gene expression, MEP pathway metabolite concentrations, and related enzyme gene expression for 1 year in the field to better understand the role of plant hormones and their long-term control. Seasonality of isoprenes was mainly driven by temperature- and light-dependent variations in substrate availability through the MEP route, as well as transcriptional and post-transcriptional control of isoprene synthase (IspS). Isoprene emissions are seasonally correlated with plant hormone levels. This was especially evident in the cytokinin profiles, which decreased in summer and increased in winter. Only 4-hydroxy-3-methylbut-2-butenyl-4-diphosphate (HMBDP) exhibited a positive connection with cytokinins among the MEP metabolites examined, suggesting that HMBDP and its biosynthetic enzyme, HMBDP synthase (HDS), play a role in channelling of MEP pathway metabolites to cytokinin production. Thus, it is probable that cytokinins have potential feed-forward regulation of isoprene production. Under long-term natural conditions, the hormonal balance of IAA/JA-Ile was not associated with IspS transcripts or isoprene emissions. This study builds on prior work by revealing differences between short- and long-term hormonal modulation of isoprene emissions in the tropical tree F. septica., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

Isoprene (2-methyl-1,3-butadiene) is emitted to the atmosphere each year in sufficient quantities to rival methane (>500 Tg C yr -1 ), primarily due to emission by trees and other plants. Chemical reactions of isoprene with other atmospheric compounds, such as hydroxyl radicals and inorganic nitrogen species (NO x ), have implications for global warming and local air quality, respectively. For many years, it has been estimated that soil-dwelling bacteria consume a significant amount of isoprene (~20 Tg C yr -1 ), but the mechanisms underlying the biological sink for isoprene have been poorly understood. Studies have indicated or confirmed the ability of diverse bacterial genera to degrade isoprene, whether by the canonical iso-type isoprene degradation pathway or through other less well-characterized mechanisms. Here, we review current knowledge of isoprene metabolism and highlight key areas for further research. In particular, examples of isoprene-degraders that do not utilize the isoprene monooxygenase have been identified in recent years. This has fascinating implications both for the mechanism of isoprene uptake by bacteria, and also for the ecology of isoprene-degraders in the environments., (© 2022 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Photolytic generation of nitric oxide (NO), isoprene, and reactive oxygen species (ROS) pre-dated life on Earth (~4 billion years ago). However, isoprene-ROS-NO interactions became relevant to climate chemistry ~50 million years ago, after aquatic and terrestrial ecosystems became dominated by isoprene-emitting diatoms and angiosperms. Today, NO and NO2 (together referred to as NOx) are dangerous biogenic gaseous atmospheric pollutants. In plants, NO, with its multiple sources and sinks, acts as a secondary messenger that regulates development at low doses and induces cell death at high doses. Likewise, biogenic isoprene is a putative antioxidant and hormone 'enabler' that hastens plant (and leaf) growth and reproduction, and improves plant tolerance to transient abiotic stresses. Using examples from controlled-chamber simulation and field studies of isoprene oxidation, we discuss the likely nature and extent of isoprene oxidation within leaves. We argue that isoprene-NO interactions vary greatly among plant species, driven by differences in isoprene emission rate and nitrate assimilation capacity (i.e. NO sink strength), ROS availability, and the within-leaf ratio between free-NO and isoprene. In a warmer and CO2-fertilized future climate, antagonism between isoprene and NO within leaves will probably occur in a NO-rich (relative to present) environment, yielding a greater proportion of isoprene oxidation products, and inducing major changes in NO-mediated growth and stress responses., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The isoprene emission rate from plants is simulated by a function of light intensity and leaf temperature, and the G-93 formula is the most extensively applied algorithm for this purpose. Isoprene is biosynthesized by the enzyme isoprene synthase (IspS), and instantly emitted from the leaf. Enzyme kinetics of IspS and substrate availability are important factors involved in the short-term leaf-level control of isoprene emissions. It is thus assumed that the parameters of G-93 may correlate with the kinetics of IspSs, however, at present there is no data available on the relationship between these two parameters. In this investigation, six IspS genes from tropical trees were cloned, their properties characterized, and the relationship between the enzyme kinetics of IspSs and the parameters of G-93 examined. There was a negative correlation between the enzyme kinetics of IspS K m and parameter C T1 of G93, which is used to define the temperature dependency of isoprene emissions. However, performance constant of IspS (k cat /K m ) only showed slight positive correlation with C T1 .suggesting that the enzyme kinetics of IspS has limited significance in controlling the temperature response of isoprene emissions. The molecular structure of IspS was further elucidated using a molecular dynamics simulation with a focus on the active site in the 6 α-helices bundle. The simulation of the enzyme-substrate complex of IspS from B. variegata predicted a new metal binding domain in helix F (E383) and catalytic motif FXRDRLXE in the A-C loop that could involve the deprotonation of dimethylallyl diphosphate (DMADP) to form a carbocation. Notably, after the binding of a metal ion and DMADP, the active-site closure mechanism was found to involve conformational alterations in the helix H-α1 and transition from a loose to tight enclosure of the 6 α-helices bundles to tune the active pocket size. The characteristics identified for the IspSs from tropical trees could help to explain regional isoprene emissions in tropical areas., (© 2022. The Author(s) under exclusive licence to The Botanical Society of Japan.)

A chemical investigation of the endophytic fungus Diaporthe destruens from the Hernandiaceae plant Illigera orbiculata C. Y. Wu collected from southern Yunnan Province, China, led to the isolation of six undescribed compounds, including two azaphilone analogs, which are a pair of epimers (13R-hydroxy-chermesinone A and 13S-hydroxy-chermesinone A); a pyrrole derivative (1-(4-(methoxymethyl)-1H-pyrrol-3-yl)ethan-1-one); an isoindolone derivative (4-hydroxy-6-methoxyisoindolin-1-one); a benzylbenzene derivative (destruensine A) and a conjectural fragment of polyketide ((2R,4R)-2-(methoxymethyl)pentane-1,4-diol) along with nine known compounds. Their structures were elucidated by spectroscopic methods and HRESIMS, and the absolute configurations were further confirmed by electronic circular dichroism (ECD) and chemical derivatization. The antimicrobial activities, anti-acetylcholinesterase activities, antiproliferation, and NO production inhibitory effects of compounds 1-15 were evaluated., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Ozone (O3) pollution affects plant growth and isoprene (ISO) emission. However, the response mechanism of isoprene emission rate (ISOrate) to elevated O3 (EO3) remains poorly understood. ISOrate was investigated in two genotypes (diploid and triploid) of Chinese white poplar (Populus tomentosa Carr.) exposed to EO3 in an open top chamber system. The triploid genotype had higher photosynthetic rate (A) and stomatal conductance (gs) than the diploid one. EO3 significantly decreased A, gs, and ISOrate of middle and lower leaves in both genotypes. In the diploid genotype, the reduction of ISOrate was caused by a systematic decrease related to ISO synthesis capacity, as indicated by decreased contents of the isoprene precursor dimethylallyl diphosphate and decreased isoprene synthase protein and activity. On the other hand, the negative effect of O3 on ISOrate of the triploid genotype did not result from inhibited ISO synthesis capacity, but from increased ISO oxidative loss within the leaf. Our findings will be useful for breeding poplar genotypes with high yield and lower ISOrate, depending on local atmospheric volatile organic compound/NOx ratio, to cope with both the rising O3 concentrations and increasing biomass demand. They can also inform the incorporation of O3 effects into process-based models of isoprene emission., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The objective of this study was to implement direct sunlight-driven conversion of CO 2 into a naturally excreted ready-to-use fuel. We engineered four different synthetic metabolic modules for biosynthesis of short-to medium-chain length hydrocarbons in the model cyanobacterium Synechocystis sp. PCC 6803. In module 1, the combination of a truncated clostridial n-butanol pathway with over-expression of the native cyanobacterial aldehyde deformylating oxygenase resulted in small quantities of propane when cultured under closed conditions. Direct conversion of CO 2 into propane was only observed in strains with CRISPRi-mediated repression of three native putative aldehyde reductases. In module 2, three different pathways towards pentane were evaluated based on the polyunsaturated fatty acid linoleic acid as an intermediate. Through combinatorial evaluation of reaction ingredients, it was concluded that linoleic acid undergoes a spontaneous non-enzymatic reaction to yield pentane and hexanal. When Synechocystis was added to the reaction, hexanal was converted into 1-hexanol, but there was no further stimulation of pentane biosynthesis even in the Synechocystis strains expressing GmLOX1. For modules 3 and 4, several different acyl-ACP thioesterases were evaluated in combination with two different decarboxylases. Small quantities of 1-heptene and 1-nonene were observed in strains expressing the desaturase-like enzyme UndB from Pseudomonas mendocina in combination with C8-C10 preferring thioesterases ('CaFatB3.5 and 'ChoFatB2.2). When UndB instead was combined with a C12-specific 'UcFatB1 thioesterase, this resulted in a ten-fold increase of alkene biosynthesis. When UndB was replaced with the light-dependent FAP decarboxylase, both undecane and tridecane accumulated, albeit with a 10-fold drop in productivity. Preliminary optimization of the RBS, promoter and gene order in some of the synthetic operons resulted in improved 1-alkene productivity, reaching a titer of 230 mg/L after 10 d with 15% carbon partitioning. In conclusion, the direct bioconversion of CO 2 into secreted and ready-to-use hydrocarbon fuel was implemented with several different metabolic systems. Optimal productivity was observed with UndB and a C12 chain-length specific thioesterase, although further optimization of the entire biosynthetic system is still possible., (Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Isoprene, a major biogenic volatile hydrocarbon of climate-relevance, indisputably mitigates abiotic stresses in emitting plants. However functional relevance of constitutive isoprene emission in unstressed plants remains contested. Isoprene and cytokinins (CKs) are synthesized from a common substrate and pathway in chloroplasts. It was postulated that isoprene emission may affect CK-metabolism. Using transgenic isoprene-emitting (IE) Arabidopsis and isoprene nonemitting (NE) RNA-interference grey poplars (paired with respective NE and IE genotypes), the life of individual IE and NE leaves from emergence to abscission was followed under stress-free conditions. We monitored plant growth rate, aboveground developmental phenotype, modelled leaf photosynthetic energy status, quantified the abundance of leaf CKs, analysed Arabidopsis and poplar leaf transcriptomes by RNA-sequencing in presence and absence of isoprene during leaf senescence. Isoprene emission by unstressed leaves enhanced the abundance of CKs (isopentenyl adenine and its precursor) by > 200%, significantly upregulated genes coding for CK-synthesis, CK-signalling and CK-degradation, hastened plant development, increased chloroplast metabolic rate, altered photosynthetic energy status, induced early leaf senescence in both Arabidopsis and poplar. IE leaves senesced sooner even in decapitated poplars where source-sink relationships and hormone homeostasis were perturbed. Constitutive isoprene emission significantly accelerates CK-led leaf and organismal development and induces early senescence independent of growth constraints. Isoprene emission provides an early-riser evolutionary advantage and shortens lifecycle duration to assist rapid diversification in unstressed emitters., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Isoprene is a small lipophilic molecule synthesized in plastids and abundantly released into the atmosphere. Isoprene-emitting plants are better protected against abiotic stresses, but the mechanism of action of isoprene is still under debate. In this study, we compared the physiological responses and proteomic profiles of Arabidopsis which express the isoprene synthase (ISPS) gene and emit isoprene with those of non-emitting plants under both drought-stress (DS) and well-watered (WW) conditions. We aimed to investigate whether isoprene-emitting plants displayed a different proteomic profile that is consistent with the metabolic changes already reported. Only ISPS DS plants were able to maintain the same photosynthesis and fresh weight of WW plants. LC-MS/MS-based proteomic analysis revealed changes in protein abundance that were dependent on the capacity for emitting isoprene in addition to those caused by the DS. The majority of the proteins changed in response to the interaction between DS and isoprene emission. These include proteins that are associated with the activation of secondary metabolisms leading to ABA, trehalose, and proline accumulations. Overall, our proteomic data suggest that isoprene exerts its protective mechanism at different levels: under drought stress, isoprene affects the abundance of chloroplast proteins, confirming a strong direct or indirect antioxidant action and also modulates signaling and hormone pathways, especially those controlling ABA synthesis. Unexpectedly, isoprene also alters membrane trafficking.

Isoprene is the most abundant non-methane hydrocarbon emitted to the atmosphere and a target of biotechnology as a source of biofuels or chemical feedstock. Measurements of the amount of isoprene or the rate of production of isoprene are important for atmospheric chemistry, evaluating biotechnology processes, and can provide information on the capacity and regulation of the methyl erythritol 4-phosphate pathway found in plants and bacteria. In this chapter we discuss techniques, and their strengths and weaknesses, of methods in common use for measuring isoprene. There are many sources of isoprene for measurements including emissions from leaves and head space analysis of reactions involving recombinant enzymes or bacterial/fungal cultures. Similarly, there are a variety of detection methods including several mass spectrometer methods that are useful for examining rates of labeling of isoprene when carbon isotopes are used., (Copyright © 2022 Elsevier Inc. All rights reserved.)

1,5-Pentanediol (1,5-PDO) is an important C5 building block for the synthesis of different value-added polyurethanes and polyesters. However, no natural metabolic pathway exists for the biosynthesis of 1,5-PDO. Herein we designed and constructed a promising nonnatural pathway for de novo production of 1,5-PDO from cheap carbohydrates. This biosynthesis route expands natural lysine pathways and employs two artificial metabolic modules to sequentially convert lysine into 5-hydroxyvalerate (5-HV) and 1,5-PDO via 5-hydroxyvaleryl-CoA. Theoretically, the 5-hydroxyvaleryl-CoA-based pathway is more energy-efficient than a recently published carboxylic acid reductase-based pathway for 1,5-PDO production. By combining strategies of systematic enzyme screening, pathway balancing, and transporter engineering, we successfully constructed a minimally engineered Escherichia coli strain capable of producing 3.19 g/L of 5-HV and 0.35 g/L of 1,5-PDO in a medium containing 20 g/L of glucose and 5 g/L lysine. Introducing the synthetic modules into a lysine producer and enhancing NADPH supply enabled the strain to accumulate 1.04 g/L of 5-HV and 0.12 g/L of 1,5-PDO using glucose as the main carbon source. This work lays the basis for the development of a biological route for 1,5-PDO production from renewable bioresources.

Amino acids are naturally occurring and structurally diverse metabolites in biological system, whose potentials for chemical expansion, however, have not been fully explored. Here, we devise a metabolic platform capable of producing industrially important C3-C5 diols from amino acids. The presented platform combines the natural catabolism of charged amino acids with a catalytically efficient and thermodynamically favorable diol formation pathway, created by expanding the substrate scope of the carboxylic acid reductase toward noncognate ω-hydroxylic acids. Using the established platform as gateways, seven different diol-convertible amino acids are converted to diols including 1,3-propanediol, 1,4-butanediol, and 1,5-pentanediol. Particularly, we afford to optimize the production of 1,4-butanediol and demonstrate the de novo production of 1,5-pentanediol from glucose, with titers reaching 1.41 and 0.97 g l -1 , respectively. Our work presents a metabolic platform that enriches the pathway repertoire for nonnatural diols with feedstock flexibility to both sugar and protein hydrolysates., Competing Interests: Competing interest statement: An invention disclosure has been filed for this work.

Finding a suitable oviposition site is a challenging task for a gravid female moth. At the same time, it is of paramount importance considering the limited capability of most caterpillars to relocate to alternative host plants. The hawkmoth, Manduca sexta (Sphingidae), oviposits on solanaceous plants. Larvae hatching on a plant that is already attacked by conspecific caterpillars can face food competition, as well as an increased exposure to predators and induced plant defenses. Here, we show that feces from conspecific caterpillars are sufficient to deter a female M. sexta from ovipositing on a plant and that this deterrence is based on the feces-emitted carboxylic acids 3-methylpentanoic acid and hexanoic acid. Using a combination of genome editing (CRISPR-Cas9), electrophysiological recordings, calcium imaging, and behavioral analyses, we demonstrate that ionotropic receptor 8a (IR8a) is essential for acid-mediated feces avoidance in ovipositing hawkmoths., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Isoprene, the ubiquitous, highly emitted non-methane volatile hydrocarbon, affects atmospheric chemistry and human health, and this makes its removal from the contaminated environment imperative. Physicochemical degradation of isoprene is inefficient and generates secondary pollutants. Therefore, biodegradation can be considered as the safer approach for its efficient abatement. This review summarizes efforts in this regard that led to tracking the diverse groups of isoprene degrading bacteria such as Methanotrophs, Xanthobacter, Nocardia, Alcaligenes, Rhodococcus, Actinobacteria, Alphaproteobacteria, Bacteriodetes, Pseudomonas, and Alcanivorax. Biodegradation of isoprene by such bacteria in batch and continuous modes has been elaborated. The products, pathways and the key enzymes associated with isoprene biodegradation have also been presented.

Isoprene (2-methyl-1,3-butadiene), the most abundantly produced biogenic volatile organic compound (BVOC) on Earth, is highly reactive and can have diverse and often detrimental atmospheric effects, which impact on climate and health. Most isoprene is produced by terrestrial plants, but (micro)algal production is important in aquatic environments, and the relative bacterial contribution remains unknown. Soils are a sink for isoprene, and bacteria that can use isoprene as a carbon and energy source have been cultivated and also identified using cultivation-independent methods from soils, leaves and coastal/marine environments. Bacteria belonging to the Actinobacteria are most frequently isolated and identified, and Proteobacteria have also been shown to degrade isoprene. In the freshwater-sediment isolate, Rhodococcus strain AD45, initial oxidation of isoprene to 1,2-epoxy-isoprene is catalyzed by a multicomponent isoprene monooxygenase encoded by the genes isoABCDEF. The resultant epoxide is converted to a glutathione conjugate by a glutathione S-transferase encoded by isoI, and further degraded by enzymes encoded by isoGHJ. Genome sequence analysis of actinobacterial isolates belonging to the genera Rhodococcus, Mycobacterium and Gordonia has revealed that isoABCDEF and isoGHIJ are linked in an operon, either on a plasmid or the chromosome. In Rhodococcus strain AD45 both isoprene and epoxy-isoprene induce a high level of transcription of 22 contiguous genes, including isoABCDEF and isoGHIJ. Sequence analysis of the isoA gene, encoding the large subunit of the oxygenase component of isoprene monooxygenase, from isolates has facilitated the development of PCR primers that are proving valuable in investigating the ecology of uncultivated isoprene-degrading bacteria.

Herbivore-induced plant volatiles play important roles in plant-insect and plant-plant interactions. The common evening primrose, Oenothera biennis, is often infested by the flea beetle, Altica oleracea, on which the predatory blue shield bug, Zicrona caerulea, is usually found. This observation suggests that the predatory bug can discriminate infested plants from intact ones to locate its prey. In this study, l-leucine-derived nitrogen-containing compounds [isovaleronitrile (3-methylbutanenitrile), (E/Z)-isovaleraldoxime and 3-methyl-1-nitrobutane] and some terpenes were identified as a characteristic volatile blend from herbivore-infested O. biennis leaves by gas chromatography/mass spectrometry, chemical synthesis, and incorporation assays using deuterium-labeled l-leucine. Volatile emission was also elicited by exogenous methyl jasmonate (MeJA), but not by mechanical damage. l-Leucine accumulated temporarily in O. biennis leaves after MeJA treatment prior to isovaleronitrile emission. Behavioral assays revealed that Z. caerulea showed a strong preference for herbivore-infested leaves, their volatiles, and isovaleronitrile in laboratory conditions.

Potassium 2-(1-hydroxypentyl)-benzoate (dl-PHPB) is a novel pro-drug of 3-n-butylphthalide (dl-NBP) that is used to treat ischemic stroke. Currently, dl-PHPB is in phase II-III clinical trials in China. In this study, we investigated the conversion and pharmacokinetics profiles of dl-PHPB in vitro and in vivo. The conversion of dl-PHPB to dl-NBP was pH- and calcium-dependent, and paraoxonase was identified as a major enzyme for the conversion in rat plasma. The pharmacokinetics, tissue distribution and excretion of dl-PHPB were studied and compared with equal-molar doses of dl-NBP in rats and dogs. The in vivo studies showed that dl-PHPB could be quickly and completely converted to dl-NBP. The plasma concentration-time course of converted dl-NBP after intravenous dl-PHPB administration was nearly the same as that after equal-molar dl-NBP. The C max and AUC of dl-NBP after oral dl-PHPB administration in rats and dogs were higher by 60% and 170%, respectively, than those after oral dl-NBP administration. Analysis of the tissue distribution of dl-PHPB revealed that converted dl-NBP was primarily distributed in fat, the brain and the stomach. In the brain, the levels of dl-NBP were relatively higher after dl-PHPB treatment by orally than after treatment with equal-molar dl-NBP. Approximately 3%-4% of dl-NBP was excreted within 72 h after dosing with dl-PHPB or dl-NBP, but no dl-PHPB was detected in urine or feces excrements. Our results demonstrate that the conversion of dl-PHPB is fast after oral or intravenous administration. Furthermore, the bioavailability of dl-PHPB was obviously better than that of dl-NBP.

Isoprene is synthesized via the chloroplastic 2- C -methyl-d-erythritol 4-phosphate/1-deoxy-d-xylulose 5-phosphate pathway (MEP/DOXP), and its synthesis is directly related to photosynthesis, except under high CO 2 concentration, when the rate of photosynthesis increases but isoprene emission decreases. Suppression of MEP/DOXP pathway activity by high CO 2 has been explained either by limited supply of the cytosolic substrate precursor, phospho enol pyruvate (PEP), into chloroplast as the result of enhanced activity of cytosolic PEP carboxylase or by limited supply of energetic and reductive equivalents. We tested the PEP-limitation hypotheses by feeding leaves with the PEP carboxylase competitive inhibitors malate and diethyl oxalacetate (DOA) in the strong isoprene emitter hybrid aspen ( Populus tremula × Populus tremuloides ). Malate feeding resulted in the inhibition of net assimilation, photosynthetic electron transport, and isoprene emission rates, but DOA feeding did not affect any of these processes except at very high application concentrations. Both malate and DOA did not alter the sensitivity of isoprene emission to high CO 2 concentration. Malate inhibition of isoprene emission was associated with enhanced chloroplastic reductive status that suppressed light reactions of photosynthesis, ultimately leading to reduced isoprene substrate dimethylallyl diphosphate pool size. Additional experiments with altered oxygen concentrations in conditions of feedback-limited and non-feedback-limited photosynthesis further indicated that changes in isoprene emission rate in control and malate-inhibited leaves were associated with changes in the share of ATP and reductive equivalent supply for isoprene synthesis. The results of this study collectively indicate that malate importantly controls the chloroplast reductive status and, thereby, affects isoprene emission, but they do not support the hypothesis that cytosolic metabolite availability alters the response of isoprene emission to changes in atmospheric composition., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Oak trees (Quercus) are hosts of diverse gall-inducing parasites, but the effects of gall formation on the physiology and biochemistry on host oak leaves is poorly understood. The influence of infection by four species from two widespread gall wasp genera, Neuroterus (N. anthracinus and N. albipes) and Cynips (C. divisa and C. quercusfolii), on foliage morphology, chemistry, photosynthetic characteristics, constitutive isoprene, and induced volatile emissions in Q. robur was investigated. Leaf dry mass per unit area (M A ), net assimilation rate per area (A A ), stomatal conductance (g s ), and constitutive isoprene emissions decreased with the severity of infection by all gall wasp species. The reduction in A A was mainly determined by reduced M A and to a lower extent by lower content of leaf nitrogen and phosphorus in gall-infected leaves. The emissions of lipoxygenase pathway volatiles increased strongly with increasing infection severity for all 4 species with the strongest emissions in major vein associated species, N. anthracinus. Monoterpene and sesquiterpene emissions were strongly elicited in N. albipes and Cynips species, but not in N. anthracinus. These results provide valuable information for diagnosing oak infections using ambient air volatile fingerprints and for predicting the impacts of infections on photosynthetic productivity and whole tree performance., (© 2017 John Wiley & Sons Ltd.)

There is a growing interest in the use of microbial fermentation for the generation of high-demand, high-purity chemicals using cheap feedstocks in an environmentally friendly manner. One example explored here is the production of isoprene (C 5 H 8 ), a hemiterpene, which is primarily polymerized to polyisoprene in synthetic rubber in tires but which can also be converted to C 10 and C 15 biofuels. The strictly anaerobic, acetogenic bacterium Clostridium ljungdahlii , used in all of the work described here, is capable of glycolysis using the Embden-Meyerhof-Parnas pathway and of carbon fixation using the Wood-Ljungdahl pathway. Clostridium - Escherichia coli shuttle plasmids, each bearing either 2 or 3 different heterologous genes of the eukaryotic mevalonic acid (MVA) pathway or eukaryotic isopentenyl pyrophosphate isomerase (Idi) and isoprene synthase (IspS), were constructed and electroporated into C. ljungdahlii These plasmids, one or two of which were introduced into the host cells, enabled the synthesis of mevalonate and of isoprene from fructose and from syngas (H 2 , CO 2 , and CO) and the conversion of mevalonate to isoprene. All of the heterologous enzymes of the MVA pathway, as well as Idi and IspS, were shown to be synthesized at high levels in C. ljungdahlii , as demonstrated by Western blotting, and were enzymatically active, as demonstrated by in vivo product synthesis. The quantities of mevalonate and isoprene produced here are far below what would be required of a commercial production strain. However, proposals are made that could enable a substantial increase in the mass yield of product formation. IMPORTANCE This study demonstrates the ability to synthesize a heterologous metabolic pathway in C. ljungdahlii , an organism capable of metabolizing either simple sugars or syngas or both together (mixotrophy). Syngas, an inexpensive source of carbon and reducing equivalents, is produced as a major component of some industrial waste gas, and it can be generated by gasification of cellulosic biowaste and of municipal solid waste. Its conversion to useful products therefore offers potential cost and environmental benefits. The ability of C. ljungdahlii to grow mixotrophically also enables the recapture, should there be sufficient reducing equivalents available, of the CO 2 released upon glycolysis, potentially increasing the mass yield of product formation. Isoprene is the simplest of the terpenoids, and so the demonstration of its production is a first step toward the synthesis of higher-value products of the terpenoid pathway., (Copyright © 2017 Diner et al.)

Isoprene is synthesized through the 2-C-methylerythritol-5-phosphate (MEP) pathway that also produces abscisic acid (ABA). Increases in foliar free ABA concentration during drought induce stomatal closure and may also alter ethylene biosynthesis. We hypothesized a role of isoprene biosynthesis in protecting plants challenged by increasing water deficit, by influencing ABA production and ethylene evolution. We performed a split-root experiment on Populus nigra L. subjected to three water treatments: well-watered (WW) plants with both root sectors kept at pot capacity, plants with both root compartments allowed to dry for 5 days (DD) and plants with one-half of the roots irrigated to pot capacity, while the other half did not receive water (WD). WD and WW plants were similar in photosynthesis, water relations, foliar ABA concentration and isoprene emission, whereas these parameters were significantly affected in DD plants: leaf isoprene emission increased despite the fact that photosynthesis declined by 85% and the ABA-glucoside/free ABA ratio decreased significantly. Enhanced isoprene biosynthesis in water-stressed poplars may have contributed to sustaining leaf ABA biosynthesis by keeping the MEP pathway active. However, this enhancement in ABA was accompanied by no change in ethylene biosynthesis, likely confirming the antagonistic role between ABA and ethylene. These results may indicate a potential cross-talk among isoprene, ABA and ethylene under drought., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Efforts were made to isolate and characterize bacteria capable of growing on methane and organic compounds, and to achieve the simultaneous degradation of more than one pollutant. Among the methanotrophs, species of Methylobacterium was able to catabolize a variety of hydrocarbons, including the branched-chain alkenes. Therefore, laboratory incubations experiments were carried out in batch mode to assess the potential of Methylobacterium sp. PV1 for degrading isoprene, the low-molecular-weight alkene, the most abundant non-methane volatile hydrocarbon present in the environment. Methylobacterium sp. PV1, isolated from paddy field soil, was characterized by pmoA and 16S rRNA gene sequencing and FAME analysis, and used for isoprene degradation. The kinetics of biodegradation is studied using the Michaelis-Menten model. The optimum degradation (80%) with maximum average relative degradation rate was observed at 150ppm isoprene. The degradation products were also analyzed using FTIR., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Taxol is an anticancer identified in both endophytic fungus and its host plant. Plant Taxol is a diterpenoid with geranylgeranyl diphosphate (GGPP) mediates the biosynthesis of its terpenoid moiety. Previous report has suggested that fungal Taxol may require terpenoid pathway for its biosynthesis. Here in this study, feeding a Taxol-producing endophytic fungus (Paraconiothyrium SSM001) with terpenoid precursors including isopentenyl pyrophosphate (IPP, isoprene) and GGPP enhanced Taxol production threefold and fivefold, respectively, compared to the control. Thus, we assumed that increasing the terpenoid pool size in particular GGPP by introducing a new copy number of GGPPS particularly from a Taxol-producing plant might increase the production level of fungal Taxol. Agrobacterium-mediated integration of Taxus canadensis geranylgeranyl diphosphate synthase (GGPPS) gene into the Paraconiothyrium SSM001 genome was successful and increased the terpenoid pool size indicated by an increase in carotenoid level and orange to red coloration of some GGPPS-transformed SSM001 colonies. Furthermore, the integration improved the level of Taxol production threefold. Feeding a GGPPS-transformed SSM001 fungus with a GGPP precursor increased the expression level of GGPPS transcript and Taxol production. The successful increase in both terpenoid and Taxol production levels due to GGPPS gene integration into the fungal genome might be a step forward in manipulating Taxol-producing endophytic fungi. Future control of the transformation time and the manipulation of the phenolic pathway could maximize the production level.

C₁ metabolism in plants is known to be involved in photorespiration, nitrogen and amino acid metabolism, as well as methylation and biosynthesis of metabolites and biopolymers. Although the flux of carbon through the C₁ pathway is thought to be large, its intermediates are difficult to measure and relatively little is known about this potentially ubiquitous pathway. In this study, we evaluated the C₁ pathway and its integration with the central metabolism using aqueous solutions of 13 C-labeled C₁ and C₂ intermediates delivered to branches of the tropical species Inga edulis via the transpiration stream. Delivery of [ 13 C]methanol and [ 13 C]formaldehyde rapidly stimulated leaf emissions of [ 13 C]methanol, [ 13 C]formaldehyde, [ 13 C]formic acid, and 13 CO₂, confirming the existence of the C1 pathway and rapid interconversion between methanol and formaldehyde. However, while [ 13 C]formate solutions stimulated emissions of 13 CO₂, emissions of [ 13 C]methanol or [ 13 C]formaldehyde were not detected, suggesting that once oxidation to formate occurs it is rapidly oxidized to CO₂ within chloroplasts. 13 C-labeling of isoprene, a known photosynthetic product, was linearly related to 13 CO₂ across C₁ and C₂ ([ 13 C₂]acetate and [2- 13 C]glycine) substrates, consistent with reassimilation of C₁, respiratory, and photorespiratory CO₂. Moreover, [ 13 C]methanol and [ 13 C]formaldehyde induced a quantitative labeling of both carbon atoms of acetic acid emissions, possibly through the rapid turnover of the chloroplastic acetyl-CoA pool via glycolate oxidation. The results support a role of the C₁ pathway to provide an alternative carbon source for glycine methylation in photorespiration, enhance CO₂ concentrations within chloroplasts, and produce key C₂ intermediates (e.g., acetyl-CoA) central to anabolic and catabolic metabolism., Competing Interests: The authors declare no conflict of interest.

Isoprene emission is a major component of biosphere-atmosphere interactions. It is the single largest source of non-methane hydrocarbon in the atmosphere. The first report of isoprene emission from plants was published in 1957 by Professor Guivi Sanadze. While humans have smelled the monoterpene hydrocarbons made by coniferous trees since their earliest migrations, only in 1957 did the world became aware that other trees make a type of hydrocarbon in even greater amounts but one to which the human nose is much less sensitive. For this 60th anniversary of the first report of isoprene emission from leaves, we trace the discovery and development of the research field, highlighting some of the most seminal observations and theoretical interpretations. This is not an exhaustive review, and many important papers are not cited, but we hope it will be of general interest to read how research in this field developed, how new observations forced us to reevaluate our theories about the significance of isoprene biosynthesis to plant physiology and adaptation and how scientific serendipity can sometimes drive a topic forward., (© 2017 John Wiley & Sons Ltd.)

Approximately one-third of volatile organic compounds (VOCs) emitted to the atmosphere consists of isoprene, originating from the terrestrial and marine biosphere, with a profound effect on atmospheric chemistry. However, isoprene provides an abundant and largely unexplored source of carbon and energy for microbes. The potential for isoprene degradation in marine and estuarine samples from the Colne Estuary, UK, was investigated using DNA-Stable Isotope Probing (DNA-SIP). Analysis at two timepoints showed the development of communities dominated by Actinobacteria including members of the genera Mycobacterium, Rhodococcus, Microbacterium and Gordonia. Representative isolates, capable of growth on isoprene as sole carbon and energy source, were obtained from marine and estuarine locations, and isoprene-degrading strains of Gordonia and Mycobacterium were characterised physiologically and their genomes were sequenced. Genes predicted to be required for isoprene metabolism, including four-component isoprene monooxygenases (IsoMO), were identified and compared with previously characterised examples. Transcriptional and activity assays of strains growing on isoprene or alternative carbon sources showed that growth on isoprene is an inducible trait requiring a specific IsoMO. This study is the first to identify active isoprene degraders in estuarine and marine environments using DNA-SIP and to characterise marine isoprene-degrading bacteria at the physiological and molecular level., (© 2017 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Isoprene (2-methyl-1,3-butadiene) is a hemiterpene molecule. It has been estimated that the plant kingdom emits 500-750 million tons of isoprene in the environment, half of which results from tropical broadleaf trees and the remainder from shrubs. Camelina (Camelina sativa (L.) Crantz) is an emerging bioenergy plant for biodiesel. In this study, we characterized isoprene formation following a diurnal/nocturnal cycle in wild-type Camelina plants. To understand the potential effects of isoprene emission on this herbaceous plant, a gray poplar Populus×canescens isoprene synthase gene (PcISPS) was overexpressed in Camelina. Transgenic plants showed increased isoprene production, and the emissions were characterized by a diurnal/nocturnal cycle. Measurements of the expression of six genes of the plastidial 2-C-methyl-d-erythriol-4-phosphate (MEP) pathway revealed that the expression patterns of three key genes were associated with isoprene formation dynamics in the three genotypic plants. Conversely, dissimilar gene expression levels existed in different genotypes, indicating that dynamics and variations occurred among plants. Moreover, transgenic plants grew shorter and developed smaller leaves than the wild-type and empty vector control transgenic plants. Photosynthetic analysis showed that the CO 2 assimilation rate, intracellular CO 2 concentration, mesophyll conductance and contents of chlorophylls a and b were similar among PcISPS transgenic, empty-vector control transgenic, and wild-type plants, indicating that the transgene did not negatively affect photosynthesis. Based on these results, we suggest that the reduced biomass was likely a trade-off consequence of the increased isoprene emission., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Instrumentation technology for metabolomics has advanced drastically in recent years in terms of sensitivity and specificity. Despite these technical advances, data analytical strategies are still in their infancy in comparison with other 'omics'. Plants are known to possess an immense diversity of secondary metabolites. Typically, more than 70% of metabolomics data are not amenable to systems biological interpretation because of poor database coverage. Here, we propose a new general strategy for mass-spectrometry-based metabolomics that incorporates all exact mass features with known sum formulas into the evaluation and interpretation of metabolomics studies. We extend the use of mass differences, commonly used for feature annotation, by redefining them as variables that reflect the remaining 'omic' domains. The strategy uses exact mass difference network analyses exemplified for the metabolomic description of two grey poplar (Populus × canescens) genotypes that differ in their capability to emit isoprene. This strategy established a direct connection between the metabotype and the non-isoprene-emitting phenotype, as mass differences pertaining to prenylation reactions were over-represented in non-isoprene-emitting poplars. Not only was the analysis of mass differences able to grasp the known chemical biology of poplar, but it also improved the interpretability of yet unknown biochemical relationships., (© 2016 John Wiley & Sons Ltd.)

Only two types of rubber oxygenases, rubber oxygenase (RoxA) and latex clearing protein (Lcp), have been described so far. RoxA proteins (RoxAs) are c -type cytochromes of ≈70 kDa produced by Gram-negative rubber-degrading bacteria, and they cleave polyisoprene into 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD), a C 15 oligo-isoprenoid, as the major end product. Lcps are common among Gram-positive rubber degraders and do not share amino acid sequence similarities with RoxAs. Furthermore, Lcps have much smaller molecular masses (≈40 kDa), are b -type cytochromes, and cleave polyisoprene to a mixture of C 20 , C 25 , C 30 , and higher oligo-isoprenoids as end products. In this article, we purified a new type of rubber oxygenase, RoxB Xsp (RoxB of Xanthomonas sp. strain 35Y). RoxB Xsp is distantly related to RoxAs and resembles RoxAs with respect to molecular mass (70.3 kDa for mature protein) and cofactor content (2 c -type hemes). However, RoxB Xsp differs from all currently known RoxAs in having a distinctive product spectrum of C 20 , C 25 , C 30 , and higher oligo-isoprenoids that has been observed only for Lcps so far. Purified RoxB Xsp revealed the highest specific activity of 4.5 U/mg (at 23°C) of all currently known rubber oxygenases and exerts a synergistic effect on the efficiency of polyisoprene cleavage by RoxA Xsp RoxB homologs were identified in several other Gram-negative rubber-degrading species, pointing to a prominent function of RoxB for the biodegradation of rubber in Gram-negative bacteria. IMPORTANCE The enzymatic cleavage of rubber (polyisoprene) is of high environmental importance given that enormous amounts of rubber waste materials are permanently released (e.g., by abrasion of tires). Research from the last decade has discovered rubber oxygenase A, RoxA, and latex clearing protein (Lcp) as being responsible for the primary enzymatic attack on the hydrophobic and water-insoluble biopolymer poly( cis -1,4-isoprene) in Gram-negative and Gram-positive rubber-degrading bacteria, respectively. Here, we provide evidence that a third type of rubber oxygenase is present in Gram-negative rubber-degrading species. Due to its characteristics, we suggest the designation RoxB for the new type of rubber oxygenase. Bioinformatic analysis of genome sequences indicates the presence of roxB homologs in other Gram-negative rubber degraders., (Copyright © 2017 American Society for Microbiology.)

Staphylococcus aureus is an important pathogen that is capable of forming biofilms on biomaterial surfaces to cause biofilm-associated infections. Autoinducer 2 (AI-2), a universal language for interspecies communication, is involved in a variety of physiological activities, although its exact role in Gram-positive bacteria, especially in S. aureus, is not yet thoroughly characterized. Herein we demonstrate that inactivation of luxS, which encodes AI-2 synthase, resulted in increased biofilm formation and higher polysaccharide intercellular adhesion (PIA) production compared with the wild-type strain in S. aureus NCTC8325. The transcript level of rbf, a positive regulator of biofilm formation, was significantly increased in the luxS mutant. All of the parental phenotypes could be restored by genetic complementation and chemically synthesized 4,5-dihydroxy-2,3-pentanedione, the AI-2 precursor molecule, suggesting that AI-2 has a signaling function to regulate rbf transcription and biofilm formation in S. aureus. Phenotypic analysis revealed that the luxS rbf double mutant produced approximately the same amount of biofilms and PIA as the rbf mutant. In addition, real-time quantitative reverse transcription-PCR analysis showed that the icaA transcript level of the rbf mutant was similar to that of the luxS rbf double mutant. These findings demonstrate that the LuxS/AI-2 system regulates PIA-dependent biofilm formation via repression of rbf expression in S. aureus. Furthermore, we demonstrated that Rbf could bind to the sarX and rbf promoters to upregulate their expression., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Marine phytoplankton emit volatile organic compounds (VOCs) such as dimethyl sulfide (DMS) and isoprene that influence air quality, cloud dynamics, and planetary albedo. We show that globally (i) marine phytoplankton taxa tend to emit either DMS or isoprene, and (ii) sea-water surface concentration and emission hotspots of DMS and isoprene have opposite latitudinal gradients. We argue that a convergence of antioxidant functions between DMS and isoprene is possible, driven by potential metabolic competition for photosynthetic substrates. Linking phytoplankton emission traits to their latitudinal niches, we hypothesize that natural selection favors DMS emission in cold (polar) waters and isoprene emission in warm (tropical) oceans, and that global warming may expand the geographic range of marine isoprene-emitters. A trade-off between DMS and isoprene at metabolic, organismal, and geographic levels may have important consequences for future marine biosphere-atmosphere interactions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Microbial communities drive many biogeochemical processes in oil sands tailings and cause greenhouse gas emissions from tailings ponds. Paraffinic solvent (primarily C 5 -C 6 ; n- and iso-alkanes) is used by some oil sands companies to aid bitumen extraction from oil sands ores. Residues of unrecovered solvent escape to tailings ponds during tailings deposition and sustain microbial metabolism. To investigate biodegradation of hydrocarbons in paraffinic solvent, mature fine tailings (MFT) collected from Albian and CNRL ponds were amended with paraffinic solvent at ~0.1wt% (final concentration: ~1000mgL -1 ) and incubated under methanogenic conditions for ~1600d. Albian and CNRL MFTs exhibited ~400 and ~800d lag phases, respectively after which n-alkanes (n-pentane and n-hexane) in the solvent were preferentially metabolized to methane over iso-alkanes in both MFTs. Among iso-alkanes, only 2-methylpentane was completely biodegraded whereas 2-methylbutane and 3-methylpentane were partially biodegraded probably through cometabolism. 16S rRNA gene pyrosequencing showed dominance of Anaerolineaceae and Methanosaetaceae in Albian MFT and Peptococcaceae and co-domination of "Candidatus Methanoregula" and Methanosaetaceae in CNRL MFT bacterial and archaeal communities, respectively, during active biodegradation of paraffinic solvent. The results are important for developing future strategies for tailings reclamation and management of greenhouse gas emissions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Rose-scented geranium (Pelargonium spp.) is one of the most important aromatic plants and is well known for its diverse perfumery uses. Its economic importance is due to presence of fragrance rich essential oil in its foliage. The essential oil is a mixture of various volatile phytochemicals which are mainly terpenes (isoprenoids) in nature. In this study, on the geranium foliage genes related to isoprenoid biosynthesis (DXS, DXR and HMGR) were isolated, cloned and confirmed by sequencing. Further, the first gene of 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, 1-deoxy-d-xylulose-5-phosphate synthase (GrDXS), was made full length by using rapid amplification of cDNA ends strategy. GrDXS contained a 2157 bp open reading frame that encoded a polypeptide of 792 amino acids having calculated molecular weight 77.5 kDa. This study is first report on heterologous expression and kinetic characterization of any gene from this economically important plant. Expression analysis of these genes was performed in different tissues as well as at different developmental stages of leaves. In response to external elicitors, such as methyl jasmonate, salicylic acid, light and wounding, all the three genes showed differential expression profiles. Further GrDXS was over expressed in the homologous (rose-scented geranium) as well as in heterologous (Withania somnifera) plant systems through genetic transformation approach. The over-expression of GrDXS led to enhanced secondary metabolites production (i.e. essential oil in rose-scented geranium and withanolides in W. somnifera). To the best of our knowledge, this is the first report showing the expression profile of the three genes related to isoprenoid biosynthesis pathways operated in rose-scented geranium as well as functional characterization study of any gene from rose-scented geranium through a genetic transformation system., (© 2016 Scandinavian Plant Physiology Society.)

Performance losses during scale-up are described since decades, but are still one of the major obstacles for industrial bioprocess development. Consequently, robustness to inhomogeneous cultivation environments is an important quality of industrial production organisms. Especially, Corynebacterium glutamicum was proven to have an outstanding resistance against rapid changes of oxygen and substrate availability as occurring in industrial scale bioreactors. This study focuses on the identification of metabolic key mechanisms for this robustness to get a deeper insight and provide future targets for process orientated strain development. A 1,5-diaminopentane producing C. glutamicum strain was cultivated in a two compartment scale-down device to create short-term environmental changes simulating industrial scale cultivation conditions. Using multi omics based methods, it is shown, that central metabolism is flexibly rearranged under short-term oxygen depletion and carbon source excess to overcome shortage in NAD + recycling. In order to balance the redox state, key enzymes for the non-oxygen dependent fermentative NAD + regeneration were significantly up-regulated while parts of non-essential pathways were down-regulated. The transfer of the cells back into the well aerated zones with low substrate concentration triggers an additional upregulation of genes for the re-assimilation of previously formed side products, showing L-lactate forming and utilizing reactions being active at the same time. Especially L-lactate as reversible and flexible external buffer for carbon and redox equivalents puts C. glutamicum in a robust position to deal with inhomogeneity in large scale processes. Biotechnol. Bioeng. 2017;114: 560-575. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Main Conclusion: Explicit proof for de novo origin of a rare post-illumination monoterpene burst and its consistency under low O 2 , shows interaction of photorespiration, photosynthesis, and isoprenoid biosynthesis during light-dark transitions. Quercus ilex L (holm oak) constitutively emits foliar monoterpenes in an isoprene-like fashion via the methyl erythritol phosphate (MEP) pathway located in chloroplasts. Isoprene-emitting plants are known to exhibit post-illumination isoprene burst, a transient emission of isoprene in darkness. An analogous post-illumination monoterpene burst (PiMB) had remained elusive and is reported here for the first time in Q. ilex. Using 13 CO 2 labelling, we show that PiMB is made from freshly fixed carbon. PiMB is rare at ambient (20%) O 2 , absent at high (50%) O 2 , and becomes consistent in leaves exposed to low (2%) O 2 . PiMB is stronger and occurs earlier at higher temperatures. We also show that primary and secondary post-illumination CO 2 bursts (PiCO 2 B) are sensitive to O 2 in Q. ilex. The primary photorespiratory PiCO 2 B is absent under both ambient and low O 2 , but is induced under high (>50%) O 2 , while the secondary PiCO 2 B (of unknown origin) is absent under ambient, but present at low and high O 2 . We propose that post-illumination recycling of photorespired CO 2 competes with the MEP pathway for photosynthetic carbon and energy, making PiMB rare under ambient O 2 and absent at high O 2 . PiMB becomes consistent when photorespiration is suppressed in Q. ilex.

Methane emissions in oil sands tailings ponds are sustained by anaerobic biodegradation of unrecovered hydrocarbons. Naphtha (primarily C 6 -C 10 ; n- iso- and cycloalkanes) is commonly used as a solvent during bitumen extraction process and its residue escapes to tailings ponds during tailings deposition. To investigate biodegradability of hydrocarbons in naphtha, mature fine tailings (MFT) collected from Albian and CNRL tailings ponds were amended with CNRL naphtha at ∼0.2 wt% (∼2000 mg L -1 ) and incubated under methanogenic conditions for ∼1600 d. Microbial communities in both MFTs started metabolizing naphtha after a lag phase of ∼100 d. Complete biodegradation/biotransformation of all n-alkanes (except partial biodegradation of n-octane in CNRL MFT) followed by major iso-alkanes (2-methylpentane, 3-methylhexane, 2- and 4-methylheptane, iso-nonanes and 2-methylnonane) and a few cycloalkanes (derivatives of cyclopentane and cyclohexane) was observed during the incubation. 16S rRNA gene pyrosequencing showed dominance of Peptococcaceae and Anaerolineaceae in Albian MFT and Anaerolineaceae and Syntrophaceae in CNRL MFT bacterial communities with co-domination of Methanosaetaceae and "Candidatus Methanoregula" in archaeal populations during active biodegradation of hydrocarbons. The findings extend the known range of hydrocarbons susceptible to methanogenic biodegradation in petroleum-impacted anaerobic environments and help refine existing kinetic model to predict greenhouse gas emissions from tailings ponds., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The lipid membrane is one of the most characteristic traits distinguishing the three domains of life. Membrane lipids of Bacteria and Eukarya are composed of fatty acids linked to glycerol-3-phosphate (G3P) via ester bonds, while those of Archaea possess isoprene-based alkyl chains linked by ether linkages to glycerol-1-phosphate (G1P), resulting in the opposite stereochemistry of the glycerol phosphate backbone. This 'lipid divide' has raised questions on the evolution of microbial life since eukaryotes are thought to have evolved from the Archaea, requiring a radical change in membrane composition. Here, we searched for homologs of enzymes involved in membrane lipid and fatty acid synthesis in a wide variety of archaeal genomes and performed phylogenomic analyses. We found that two uncultured archaeal groups, i.e. marine euryarchaeota group II/III and 'Lokiarchaeota', recently discovered descendants of the archaeal ancestor leading to eukaryotes, lack the gene to synthesize G1P and, consequently, the capacity to synthesize archaeal membrane lipids. However, our analyses reveal their genetic capacity to synthesize G3P-based 'chimeric lipids' with either two ether-bound isoprenoidal chains or with an ester-bound fatty acid instead of an ether-bound isoprenoid. These archaea may reflect the 'archaea-to-eukaryote' membrane transition stage which have led to the current 'lipid divide'., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The release of stress-driven volatiles throughout leaf development has been little studied. Therefore, we subjected poplar leaves during their developmental stage (from 2 days to 2 weeks old) to wounding by a single punch hole, and measured online the wound-induced volatile organic compound emissions. Our study shows that the emission of certain volatile compounds fades with increasing leaf age. Among these compounds we found lipoxygenase products (LOX products), acetaldehyde, methyl benzoate, methyl salicylate, and mono- and sesquiterpenes. In parallel, we studied the fading of constitutive emissions of methanol during leaf maturation, as well as the rise in isoprene constitutive emission during leaf maturation and its relationship to leaf photosynthetic capacity. We found highly significant relationships between leaf chlorophyll content, photosynthetic capacity, and leaf size during leaf ageing. As the level of constitutive defences increases with increasing leaf age, the strength of the volatile signal is expected to be gradually reduced. The higher elicitation of volatile organic compound emissions (especially LOX products) in younger leaves could be an evolutionary defence against herbivory, given that younger leaves are usually more subjected to infestation and herbivory.

Current studies on microbial isoprene biosynthesis have mostly focused on regulation of the upstream mevalonic acid (MVA) or methyl-erythritol-4-phosphate (MEP) pathway. However, the downstream bottleneck restricting isoprene biosynthesis capacity caused by the weak expression and low activity of plant isoprene synthase (ISPS) under microbial fermentation conditions remains to be alleviated. Here, based on a previously constructed Saccharomyces cerevisiae strain with enhanced precursor supply, we strengthened the downstream pathway through increasing both the expression and activity of ISPS to further improve isoprene production. Firstly, a two-level expression enhancement system was developed for the P GAL1 -controlled ISPS by overexpression of GAL 4. Meanwhile, the native GAL1/7/10 promoters were deleted to avoid competition for the transcriptional activator Gal4p, and GAL80 was disrupted to eliminate the dependency of gene expression on galactose induction. The IspS expression was obviously elevated upon enhanced Gal4p supply, and the isoprene production was improved from 6.0mg/L to 23.6mg/L in sealed-vial cultures with sucrose as carbon source. Subsequently, a novel high-throughput screening method was developed based on precursor toxicity and used for ISPS directed evolution towards enhanced catalytic activity. Combinatorial mutagenesis of the resulting ISPS mutants generated the best mutant ISPSM4, introduction of which into the GAL4-overexpressing strain YXM29 achieved 50.2mg/L of isoprene in sealed vials, and the isoprene production reached 640mg/L and 3.7g/L in aerobic batch and fed-batch fermentations, respectively. These results demonstrated the effectiveness of the proposed combinatorial engineering strategy in isoprene biosynthesis, which might also be feasible and instructive for biotechnological production of other valuable chemicals., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Background: Isoprene, a volatile C5 hydrocarbon, is an important platform chemical used in the manufacturing of synthetic rubber for tires and various other applications, such as elastomers and adhesives., Results: In this study, Escherichia coli MG1655 harboring Populus trichocarpa isoprene synthase (PtispS) and the exogenous mevalonate (MVA) pathway produced 80 mg/L isoprene. Codon optimization and optimal expression of the ispS gene via adjustment of the RBS strength and inducer concentration increased isoprene production to 199 and 337 mg/L, respectively. To augment expression of MVA pathway genes, the MVA pathway was cloned on a high-copy plasmid (pBR322 origin) with a strong promoter (P trc ), which resulted in an additional increase in isoprene production up to 956 mg/L. To reduce the formation of byproducts derived from acetyl-CoA (an initial substrate of the MVA pathway), nine relevant genes were deleted to generate the E. coli AceCo strain (E. coli MG1655 ΔackA-pta, poxB, ldhA, dld, adhE, pps, and atoDA). The AceCo strain harboring the ispS gene and MVA pathway showed enhanced isoprene production of 1832 mg/L in flask culture with reduced accumulation of byproducts., Conclusions: We achieved a 23-fold increase in isoprene production by codon optimization of PtispS, augmentation of the MVA pathway, and deletion of genes involved in byproduct formation.

Plant isoprene emissions respond to light and temperature similarly to photosynthesis, but CO 2 dependencies of isoprene emission and photosynthesis are profoundly different, with photosynthesis increasing and isoprene emission decreasing with increasing CO 2 concentration due to reasons not yet understood. We studied isoprene emission, net assimilation rate, and chlorophyll fluorescence under different CO 2 and O 2 concentrations in the strong isoprene emitter hybrid aspen (Populus tremula × Populus tremuloides), and used rapid changes in ambient CO 2 or O 2 concentrations or light level to induce oscillations. As isoprene-emitting species support very high steady-state chloroplastic pool sizes of the primary isoprene substrate, dimethylallyl diphosphate (DMADP), which can mask the effects of oscillatory dynamics on isoprene emission, the size of the DMADP pool was experimentally reduced by either partial inhibition of isoprenoid synthesis pathway by fosmidomycin-feeding or by changes in ambient gas concentrations leading to DMADP pool depletion in intact leaves. In feedback-limited conditions observed at low O 2 and/or high CO 2 concentration under which the rate of photosynthesis is governed by the limited rate of ATP and NADPH formation due to low chloroplastic phosphate levels, oscillations in photosynthesis and isoprene emission were repeatedly induced by rapid environmental modifications in both partly fosmidomycin-inhibited leaves and in intact leaves with in vivo reduced DMADP pools. The oscillations in net assimilation rate and isoprene emission in feedback-inhibited leaves were in the same phase, and relative changes in the pools of photosynthetic metabolites and DMADP estimated by in vivo kinetic methods were directly proportional through all oscillations induced by different environmental perturbations. We conclude that the oscillations in isoprene emission provide direct experimental evidence demonstrating that the response of isoprene emission to changes in ambient gas concentrations is controlled by the chloroplastic reductant supply., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Plant isoprene emissions have been linked to several reaction pathways involved in atmospheric photochemistry. Evidence exists from a limited set of past observations that isoprene emission rate (I s ) decreases as a function of increasing atmospheric CO 2 concentration, and that increased temperature suppresses the CO 2 effect. We studied interactions between intercellular CO 2 concentration (C i ) and temperature as they affect I s in field-grown hybrid poplar trees in one of the warmest climates on earth - the Sonoran Desert of the southwestern United States. We observed an unexpected midsummer downregulation of I s despite the persistence of relatively high temperatures. High temperature suppression of the I s :C i relation occurred at all times during the growing season, but sensitivity of I s to increased C i was greatest during the midsummer period when I s was lowest. We interpret the seasonal downregulation of I s and increased sensitivity of I s to C i as being caused by weather changes associated with the onset of a regional monsoon system. Our observations on the temperature suppression of the I s :C i relation are best explained by the existence of a small pool of chloroplastic inorganic phosphate, balanced by several large, connected metabolic fluxes, which together, determine the C i and temperature dependencies of phosphoenolpyruvate import into the chloroplast., (© 2016 John Wiley & Sons Ltd.)

Physiological, biochemical and morpho-anatomical traits that determine the phenotypic plasticity of plants under drought were tested in two Arundinoideae with contrasting habitats, growth traits and metabolism: the fast-growing Arundo donax, which also is a strong isoprene emitter, and the slow-growing Hakonechloa macra that does not invest on isoprene biosynthesis. In control conditions, A. donax displayed not only higher photosynthesis but also higher concentration of carotenoids and lower phenylpropanoid content than H. macra. In drought-stressed plants, photosynthesis was similarly inhibited in both species, but substantially recovered only in A. donax after rewatering. Decline of photochemical and biochemical parameters, increased concentration of CO2 inside leaves, and impairment of chloroplast ultrastructure were only observed in H. macra indicating damage of photosynthetic machinery under drought. It is suggested that volatile and non-volatile isoprenoids produced by A. donax efficiently preserve the chloroplasts from transient drought damage, while H. macra invests on phenylpropanoids that are less efficient in preserving photosynthesis but likely offer better antioxidant protection under prolonged stress., (© 2016 John Wiley & Sons Ltd.)

Isoprene emission from plants is very sensitive to environmental temperature both at short-term and long-term scales. Our previous study demonstrated suppression of isoprene emission by cold temperatures in a high emitting tropical tree Ficus septica and revealed a strong correlation of emission to isoprene synthase (IspS) protein levels. When challenged with decreasing daily temperatures from 30 to 12 °C, F. septica completely stopped isoprene emission at 12 °C, only to recover on the second day after re-exposure to 30 °C. Here, we explored this regulation of isoprene emission in response to environmental temperature by a comprehensive analysis of transcriptome data, gene expressions and metabolite pools of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. MEP pathway genes and metabolites dynamics did not support substrate-level limitations as major control over observed basal emission, but transcriptome data, network inferences and putative regulatory elements on IspS promoter suggested transcriptional regulation of IspS gene through circadian rhythm and phytohormone signalling processes. Expression levels of 29 genes involved in these pathways were examined by quantitative real-time PCR. We propose that temperature controls over basal isoprene emission at a time-scale of hours to few days are regulated by phytohormone-mediated transcriptional modulation of IspS gene under synchronization by the circadian clock., (© 2016 John Wiley & Sons Ltd.)

Co-metabolic bioremediation is supposed to be an impressive and promising approach in the elimination technology of methyl tert-butyl ether (MTBE), which was found to be a common pollutant worldwide in the ground or underground water in recent years. In this paper, bacterial strain DZ13 (which can co-metabolically degrade MTBE) was isolated and named as Pseudomonas sp. DZ13 based on the result of 16S rRNA gene sequencing analysis. Strain DZ13 could grow on n-alkanes (C₅-C₈), accompanied with the co-metabolic degradation of MTBE. Diverse n-alkanes with different carbon number showed a significant influence on the degradation rate of MTBE and accumulation of tert-butyl alcohol (TBA). When Pseudomonas sp. DZ13 co-metabolically degraded MTBE with n-pentane as the growth substrate, a higher MTBE-degrading rate (Vmax = 38.1 nmol/min/mgprotein, Ks = 6.8 mmol/L) and lower TBA-accumulation was observed. In the continuous degradation experiment, the removal efficiency of MTBE by Pseudomonas sp. Strain DZ13 did not show an obvious decrease after five times of continuous addition., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.