The potency of mitochondria enlargement for mitochondria-mediated terpenoid production in yeast.

Terpenoids are widely used in the food, beverage, cosmetics, and pharmaceutical industries. Microorganisms have been extensively studied for terpenoid production. In yeast, the introduction of the mevalonate (MVA) pathway in organelles in addition to the augmentation of its own MVA pathway have been challenging. Introduction of the MVA pathway into mitochondria is considered a promising approach for terpenoid production because acetyl-CoA, the starting molecule of the MVA pathway, is abundant in mitochondria. However, mitochondria comprise only a small percentage of the entire cell. Therefore, we hypothesized that increasing the total mitochondrial volume per cell would increase terpenoid production. First, we ascertained that the amounts of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the final molecules of the MVA pathway, were 15-fold higher of the strain expressing the MVA pathway in mitochondria than in the wild-type yeast strain. Second, we found that different deletion mutants induced different mitochondrial volumes by measuring the mitochondrial volume in various deletion mutants affecting mitochondrial morphology; for example,Δmdm32 increased mitochondrial volume, and Δfzo1 decreased it. Finally, the effects of mitochondrial volume on amounts of IPP/DMAPP and terpenoids (squalene or ß-carotene) were investigated using mutants harboring large or small mitochondria expressing the MVA pathway in mitochondria. Amounts of IPP/DMAPP and terpenoids (squalene or ß-carotene) increased when the mitochondrial volume expanded. Introducing the MVA pathway into mitochondria for terpenoid production in yeast may become more attractive by enlarging the mitochondrial volume. KEY POINTS: • IPP/DMAPP content increased in the strain expressing the MVA pathway in mitochondria • IPP/DMAPP and terpenoid contents are positively correlated with mitochondrial volume • Enlarging the mitochondria may improve mitochondria-mediated terpenoid production.

The isoprene-responsive phosphoproteome provides new insights into the putative signalling pathways and novel roles of isoprene.

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.

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.

Relationship between seasonal variation in isoprene emission and plant hormone profiles in the tropical plant Ficus septica.

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.

Automatization of metabolite extraction for high-throughput metabolomics: case study on transgenic isoprene-emitting birch.

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.

Identification and functional analysis of isopentenyl pyrophosphate isomerase genes in the whiteflies Bemisia tabaci (Hemiptera: Aleyrodidae).

The juvenile hormone (JH) plays a vital role in the regulation of a number of physiological processes, including development, reproduction, and ovarian maturation. Isopentenyl pyrophosphate isomerase (IPPI) is a key enzyme in the biosynthetic pathway of JH. In this study, we identified an isopentenyl pyrophosphate isomerase protein from Bemisia tabaci and named it BtabIPPI. The open reading frame (ORF) of BtabIPPI is 768 bp and encodes a protein of 255 amino acids that contains a conserved domain of the Nudix family. The temporal and spatial expression profiles showed that BtabIPPI was highly expressed in the female adults.RNA interference (RNAi)-mediated silencing of BtabIPPI reduced JH titers and the relative expression of vitellogenin receptor (VgR) and JH signaling pathway genes, resulting in a dramatic reduction in fecundity and hatchability. These results indicate that the BtabIPPI gene plays an important role in the female fecundity of B. tabaci. This study will broaden our understanding of the function of IPPI in regulating insect reproduction and provide a theoretical basis for targeting IPPI for pest control in the future.

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

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

Analysis of Essential Isoprene Metabolic Pathway Proteins in Variovorax sp. Strain WS11.

Isoprene monooxygenase (IsoMO, encoded by isoABCDEF) initiates the oxidation of the climate-active gas isoprene, with the genes isoGHIJ and aldH nearly always found adjacent to isoABCDEF in extant and metagenome-derived isoprene degraders. The roles of isoGHIJ and aldH are uncertain, although each is essential to isoprene degradation. We report here the characterization of these proteins from two model isoprene degraders, Rhodococcus sp. strain AD45 and Variovorax sp. strain WS11. The genes isoHIJ and aldH from Variovorax and aldH from Rhodococcus were expressed individually in Escherichia coli as maltose binding protein fusions to overcome issues of insolubility. The activity of two glutathione S-transferases from Variovorax, IsoI and IsoJ was assessed with model substrates, and the conversion of epoxyisoprene to the intermediate 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB) was demonstrated. The next step of the isoprene metabolic pathway of Variovorax is catalyzed by the dehydrogenase IsoH, resulting in the conversion of HGMB to 2-glutathionyl-2-methyl-3-butenoic acid (GMBA). The aldehyde dehydrogenases (AldH) from Variovorax and Rhodococcus were examined with a variety of aldehydes, with both exhibiting maximum activity with butanal. AldH significantly increased the rate of production of NADH when added to the IsoH-catalyzed conversion of HGMB to GMBA (via GMB), suggesting a synergistic role for AldH in the isoprene metabolic pathway. An in silico analysis of IsoG revealed that this protein, which is essential for isoprene metabolism in Variovorax, is an enzyme of the formyl CoA-transferase family and is predicted to catalyze the formation of a GMBA-CoA thioester as an intermediate in the isoprene oxidation pathway. IMPORTANCE Isoprene is a climate-active gas, largely produced by trees, which is released from the biosphere in amounts equivalent to those of methane and all other volatile organic compounds combined. Bacteria found in many environments, including soils and on the surface of leaves of isoprene-producing trees, can grow on isoprene and thus may represent a significant biological sink for this globally significant volatile compound and remove isoprene before it escapes to the atmosphere, thus reducing its potency as a climate-active gas. The initial oxidation of isoprene by bacteria is mediated by isoprene monooxygenase encoded by the genes isoABCDEF. In isoprene-degrading bacteria, a second gene cluster, isoGHIJ, is also present, although the exact role in isoprene degradation by the proteins encoded by these genes is uncertain. This investigation sheds new light on the roles of these proteins in the isoprene oxidation pathway in two model isoprene-degrading bacteria of the genera Rhodococcus and Variovorax.

Peering down the sink: A review of isoprene metabolism by bacteria.

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 (NOx ), 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.

Origin, evolution, and future of isoprene and nitric oxide interactions within leaves.

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.

Membrane Permeant Analogs for Independent Cellular Introduction of the Terpene Precursors Isopentenyl- and Dimethylallyl-Pyrophosphate.

Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the central five-carbon precursors to all terpenes. Despite their significance, exogenous, independent delivery of IPP and DMAPP to cells is impossible as the negatively charged pyrophosphate makes these molecules membrane impermeant. Herein, we demonstrate a facile method to circumvent this challenge through esterification of the ß-phosphate with two self-immolative esters (SIEs) that neutralize the negatively charged pyrophosphate to yield membrane-permeant analogs of IPP and DMAPP. Following cellular incorporation, general esterase activity initiates cleavage of the SIEs, resulting in traceless release of IPP and DMAPP for metabolic utilization. Addition of the synthesized IPP and DMAPP precursor analogs rescued cell growth of glioblastoma (U-87MG) cancer cells concurrently treated with the HMG-CoA reductase inhibitor pitavastatin, which otherwise abrogates cell growth via blocking production of IPP and DMAPP. This work demonstrates a new application of a prodrug strategy to incorporate a metabolic intermediate and promises to enable future interrogation of the distinct biological roles of IPP and DMAPP.

Molecular characteristics of isoprene synthase and its control effects on isoprene emissions from tropical trees.

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 Km and parameter CT1 of G93, which is used to define the temperature dependency of isoprene emissions. However, performance constant of IspS (kcat/Km) only showed slight positive correlation with CT1.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.

Isoprene measurements to assess plant hydrocarbon emissions and the methylerythritol pathway.

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.

Metabolic Engineering of the Isopentenol Utilization Pathway Enhanced the Production of Terpenoids in Chlamydomonas reinhardtii.

Eukaryotic green microalgae show considerable promise for the sustainable light-driven biosynthesis of high-value fine chemicals, especially terpenoids because of their fast and inexpensive phototrophic growth. Here, the novel isopentenol utilization pathway (IUP) was introduced into Chlamydomonas reinhardtii to enhance the hemiterpene (isopentenyl pyrophosphate, IPP) titers. Then, diphosphate isomerase (IDI) and limonene synthase (MsLS) were further inserted for limonene production. Transgenic algae showed 8.6-fold increase in IPP compared with the wild type, and 23-fold increase in limonene production compared with a single MsLS expressing strain. Following the culture optimization, the highest limonene production reached 117 µg/L, when the strain was cultured in a opt2 medium supplemented with 10 mM isoprenol under a light: dark regimen. This demonstrates that transgenic algae expressing the IUP represent an ideal chassis for the high-value terpenoid production. The IUP will facilitate further the metabolic and enzyme engineering to enhance the terpenoid titers by significantly reducing the number of enzyme steps required for an optimal biosynthesis.

'Omics-guided prediction of the pathway for metabolism of isoprene by Variovorax sp. WS11.

Bacteria that inhabit soils and the leaves of trees partially mitigate the release of the abundant volatile organic compound, isoprene (2-methyl-1,3-butadiene). While the initial steps of isoprene metabolism were identified in Rhodococcus sp. AD45 two decades ago, the isoprene metabolic pathway still remains largely undefined. Limited understanding of the functions of isoG, isoJ and aldH and uncertainty in the route of isoprene-derived carbon into central metabolism have hindered our understanding of isoprene metabolism. These previously uncharacterised iso genes are essential in Variovorax sp. WS11, determined by targeted mutagenesis. Using combined 'omics-based approaches, we propose the complete isoprene metabolic pathway. Isoprene is converted to propionyl-CoA, which is assimilated by the chromosomally encoded methylmalonyl-CoA pathway, requiring biotin and vitamin B12, with the plasmid-encoded methylcitrate pathway potentially providing robustness against limitations in these vitamins. Key components of this pathway were induced by both isoprene and its initial oxidation product, epoxyisoprene, the principal inducer of isoprene metabolism in both Variovorax sp. WS11 and Rhodococcus sp. AD45. Analysis of the genomes of distinct isoprene-degrading bacteria indicated that all of the genetic components of the methylcitrate and methylmalonyl-CoA pathways are not always present in isoprene degraders, although incorporation of isoprene-derived carbon via propionyl-CoA and acetyl-CoA is universally indicated.

Elevated ozone inhibits isoprene emission of a diploid and a triploid genotype of Populus tomentosa by different mechanisms.

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.

MICROBIAL isoprene production: an overview.

Isoprene, a volatile C5 hydrocarbon, is a precursor of synthetic rubber and an important building block for a variety of natural products, solely being produced by petrochemical routes. To mitigate the ever-increasing contribution of petrochemical industry to global warming through significant carbon (CO2) evolution, bio-based process for isoprene production using microbial cell factories have been explored. Highly efficient fermentation-based processes have been studied for little over a decade now with extensive research on the rational strain development for creating robust strains for commercial isoprene production. Most of these studies involved sugars as feedstocks and using naturally occurring isoprene pathways viz., mevalonate and methyl erythritol pathway in E. coli. Recent advances, driven by efforts in reducing environmental pollution, have focused on utilization of inorganic CO2 by cyanobacteria or syngas from waste gases by acetogens for isoprene production. This review endeavors to capture the latest relevant progress made in rational strain development, metabolic engineering and synthetic biology strategies used, challenges in fermentation process development at lab and commercial scale production of isoprene along with a future perspective pertaining to this area of research.

Isoprene Emission Influences the Proteomic Profile of Arabidopsis Plants under Well-Watered and Drought-Stress Conditions.

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.

Purification and Characterization of the Isoprene Monooxygenase from Rhodococcus sp. Strain AD45.

Isoprene (2-methyl-1,3-butadiene) is a climate-active gas released to the atmosphere in large quantities, comparable to methane in magnitude. Several bacteria have been isolated which can grow on isoprene as a sole carbon and energy source, but very little information is available about the degradation of isoprene by these bacteria at the biochemical level. Isoprene utilization is dependent on a multistep pathway, with the first step being the oxidation of isoprene to epoxy-isoprene. This is catalyzed by a four-component soluble diiron monooxygenase, isoprene monooxygenase (IsoMO). IsoMO is a six-protein complex comprising an oxygenase (IsoABE), containing the di-iron active site, a Rieske-type ferredoxin (IsoC), a NADH reductase (IsoF), and a coupling/effector protein (IsoD), homologous to the soluble methane monooxygenase and alkene/aromatic monooxygenases. Here, we describe the purification of the IsoMO components from Rhodococcus sp. AD45 and reconstitution of isoprene-oxidation activity in vitro. Some IsoMO components were expressed and purified from the homologous host Rhodococcus sp. AD45-ID, a Rhodococcus sp. AD45 strain lacking the megaplasmid which contains the isoprene metabolic gene cluster. Others were expressed in Escherichia coli and purified as fusion proteins. We describe the characterization of these purified components and demonstrate their activity when combined with Rhodococcus sp. AD45 cell lysate. Demonstration of IsoMO activity in vitro provides a platform for further biochemical and biophysical characterization of this novel soluble diiron center monooxygenase, facilitating new insights into the enzymatic basis for the bacterial degradation of isoprene. IMPORTANCE Isoprene is a highly abundant climate-active gas and a carbon source for some bacteria. Analyses of the genes encoding isoprene monooxygenase (IsoMO) indicate this enzyme is a soluble diiron center monooxygenase in the same family of oxygenases as soluble methane monooxygenase, alkene monooxygenase, and toluene monooxygenase. We report the initial biochemical characterization of IsoMO from Rhodococcus, the first from any bacterium, describing the challenging purification and reconstitution of in vitro activity of its four components. This study lays the foundation for future detailed mechanistic studies of IsoMO, a key enzyme in the global isoprene cycle.