Remodeling Isoprene Pyrophosphate Metabolism for Promoting Terpenoids Bioproduction

Terpenoids are the largest family of natural products.They are made from the building block isoprene pyrophosphate(IPP),and their bioproduction using engineered cell factories has received a great deal of attention.To date,the insufficient metabolic supply of IPP remains a great challenge for the efficient synthesis of terpenoids.In this work,we discover that the imbalanced metabolic flux distribution between the central metabolism and the IPP supply hinders IPP accumulation in Bacillus subtilis(B.subtilis).Therefore,we remodel the IPP metabolism using a series of genetically encoded two-input-multioutput(TIMO)circuits that are responsive to pyruvate or/and malonyl-CoA,resulting in an IPP pool that is significantly increased by up to four-fold.As a proof-of-concept validation,we design an IPP metabolism remodeling strategy to improve the production of three valuable terpenoids,including menaquinone-7(MK-7,4.1-fold),lycopene(9-fold),andβ-carotene(0.9-fold).In particular,the titer of MK-7 in a 50-L bioreactor reached 1549.6 mg·L^(-1),representing the highest titer reported so far.Thus,we propose a TIMO genetic circuits-assisted IPP metabolism remodeling framework that can be generally used for the synergistic fine-tuning of complicated metabolic modules to achieve the efficient bioproduction of terpenoids.

A HIGHLY ACTIVE NEODYMIUM CHLORIDE ISOPROPANOL COMPLEX/MODIFIED METHYLALUMINOXANE CATALYST FOR PREPARING POLYISOPRENE WITH HIGH CIS-1,4 STEREOSPECIFICITY AND NARROW MOLECULAR WEIGHT DISTRIBUTION

Neodymium chloride isopropanol complex （NdCl3.3＇prOH） activated by modified methylaluminoxane （MMAO） was examined in isoprene polymerization in hexane, with regards to Nd compounds, aluminum （Al） compounds, [Al]/[Nd] ratio, polymerization temperature and time. NdCl3＇3iprOH exhibited high activity producing polymers feasting high cis-l,4 stereospecificity （〉 96%）, very high molecular weight （Mn 〉 1.0 × 10^6） and fairly narrow molecular weight distribution （MWD, Mw/Mn 〈 2.0） simultaneously. In comparison, neodymium isopropoxide also showed high activity providing polymers with narrow MWD （Mw/Mn = 2.07）, but somewhat low cis-1,4 content （ca. 92%）, while neodymium chloride had no activity under present polymerization conditions. The Al compounds affected the polymer yield in the order of Al（i-Bu）3 〉 MMAO 〉 Al（i-Bu）2H. MMAO as cocatalyst afforded polyisoprene with high Mn over 1.0 × 10^6, whereas as stronger chain transfer agent than MMAO, AI（i-Bu）3 and AI（i-Bu）EH yielded polymers with low Mn （1.0 × 10^5-8.0 × 10^5）. NdCl3·3＇PrOH/MMAO catalyst showed a fairly good catalytic activity even at relatively low [Al]/[Nd] ratio of 30, and the produced polymer remained high cis-1,4 content of 95.8% along with high Mn over 1.0× 10^6 even at elevated temperatures up to 70℃. The polymerization rate is of the first order with respect to the concentration of isoprene. The mechanism of active species formation was discussed preliminarily.

Polymerization of isoprene catalyzed by neodymium heterocyclic Schiff base complex

Neodymium-based heterocyclic Schiff base complex was prepared and applied for the coordination polymerization of isoprene. This complex polymerized isoprene to afford products featuring high cis-1,4 stereospecificity （ca. 95%） and high molecular weight （ca, 10^5） in the presence of the triisobutyl aluminium （AliBu3） as cocatalyst, The microstructure of obtained polyisoprene was investigated by FTIR, 1^H NMR. Two different kinds of active centers in the catalyst system were examined by GPC method.

IN SITU MONITORING OF COORDINATION COPOLYMERIZATION OF BUTADIENE AND ISOPRENE VIA ATR-FTIR SPECTROSCOPY

FTIR spectroscopy in combination with a diamond tipped attenuated total reflectance （ATR） immersion probe was utilized to study in situ the copolymerization of butadiene （Bd） and isoprene （Ip） with neodymium-based catalyst in hexane. The relationship between the signal intensity of monomer and its concentration was investigated. The kinetic study of copolymerization of Bd and Ip was further conducted, and the monomer reactivity ratios were determined via in situ ATR FTIR. The signal band at 1010 cm^-1 was assigned to wagging vibration of Bd and its intensity was proportional to Bd concentration （[Bd]） in the range of 0.46-3.88 mol.L^-1. The signal bands at 890 and 989 cm^-1 were assigned to wagging vibration of Ip and the signal intensity was also proportional to Ip concentration （[Ip]） in the range of 0.08-4.73 mol·L^-1 at 890 cm^-1 and 0.08-7.49 mol·L^-1 at 989 cm^-1, respectively. Thus the signal band at 1010 cm^-1 was chosen to monitor Bd concentration and bands at 989 and 890 cm^-1 to monitor Ip concentration during the copolymerization, respectively. It was demonstrated that the conversions of Bd and Ip calculated from FTIR data agreed very well with those obtained gravimetrically. The poiymerization rates were first order with respect to both [Bd] and [Ip], respectively at different polymerization temperatures. The apparent propagation activation energy for Bd and Ip could be determined to be 54.4 kJ·mol^-1 and 57.7 kJ·mol^-1, respectively. The monomer reactivity ratios were calculated to be 1.08 for Bd （rBd） and 0.48 for IP （rIp） based on FTIR data. The Bd-Ip copolymer products with random sequence could be obtained with only one glass transition temperature.

Biogenic isoprene emissions over China:sensitivity to the CO2 inhibition effect

Isoprene emissions emitted from vegetation are one of the most important precursors for tropospheric ozone and secondary organic aerosol formation. The authors estimate the biogenic isoprene emissions in China over 2006-2011 using a global chemical transport model （GEOS- Chem） driven by meteorological fields from the assimilated meteorological data from MERRA. The authors incorporate three different parameterizations of isoprene-CO2 interaction into the model, and perform three sensitivity simulations to investigate the effect of CO2 inhibition on isoprene emissions for the period 2006-2011 in China. The annual isoprene emissions rate across China is simulated to be 12.62 Tg C yr-1, averaged over 2006-2011, and decreases by about 2.7%-7.4% when the CO2 inhibition schemes are included. The CO2 inhibition effect might be significant in regions where the CO2 concentration and isoprene emissions are high. Estimates of isoprene emissions can differ depending on the scheme of CO2 inhibition. According to the results obtained from the sensitivity simulations, the authors find that the CO2 inhibition effect leads to 5.6% ±2.3% reductions in annual isoprene emissions over China. The authors also find that inclusion of CO2 inhibition can substantially alter the sensitivity of isoprene emissions to the changes in meteorological conditions during the study period.

Photo-oxidation of Isoprene with Organic Seed： Estimates of Aerosol Size Distributions Evolution and Formation Rates

Indoor smog chamber experiments have been conducted to investigate the dynamics of sec- ondary organic aerosol （SOA） formation from OH-initiated photo-oxidation of isoprene in the presence of organic seed aerosol. The dependence of the size distributions of SOA on both the level of pre-existing particles generated in situ from the photo-oxidation of trace hydrocarbons of indoor atmosphere and the concentration of precursor, has been investi- gated. It was shown that in the presence of high-level seed aerosol and low-level isoprene （typical urban atmospheric conditions）, particle growth due to condensation of secondary organic products on pre-existing particles dominated; while in the presence of low-level seed aerosol and comparatively high-level isoprene （typical atmospheric conditions in rural re-gion）, bimodal structures appeared in the size distributions of SOA, which corresponded to new particle formation resulting from homogeneous nucleation and particle growth due to condensation of secondary organic products on the per-existing particles respectively. The effects of concentrations of organic seed particles on SOA were also investigated. The particle size distributions evolutions as well as the corresponding formation rates of new particles in different conditions were also estimated.

Biogenic Isoprene Emission Mechanism from ^(13)CO_2 Exposure Experiments

Biogenic isoprene emissions have been believed to be from only photosynthesis processes in plant. However nocturnal isoprene emission from pine is detected. And by feeding 13CO2 to plants, it is found that both photosynthesis pathway and light independent processes contribute to isoprene emissions.

Quantum chemical descriptors based QSAR modeling of neodymium carboxylate catalysts for coordination polymerization of isoprene

Based on the quantum chemical descriptors and the activities for isoprene polymerization of 12 neodymium carboxylates obtained by authors’earlier work,statistical analysis of data was made and a QSAR model correlating the quantum chemical descriptors and the activity was built with the partial least square(PLS)approach.The model is:A=51602εHOMO+6σ+12546,which indicates that the catalytic activity A is positively correlated with the HOMO energyεHOMOand the ligand polarizabilityσ,with the contribution ofσbeing larger,εHOMO smaller.The model’s coefficient of determination r^2=0.96 and that of cross validation q^2=0.94,both being close to 1,which means that its quality is well and its predictive power is strong.Analysis of the modeling process and the resulted QSAR model,together with the interpretation of the model’s mechanism,also shows that the model obtained from this study is valid and reliable.According to the QSAR model,the mechanism of catalyst activity can be interpreted as that neodymium carboxylates with higher HOMO energy and larger ligand polarizability are easier to react with the co-catalysts so as to produce more active and stable centers of catalyst,resulting in a higher activity.

Biogenic Isoprene and Its Impact on Human Health in Dependence on Meteorological Conditions

Urban green areas have an important implication on the local climate. A cross-linkage of many small green spaces could result in decreasing the effect of the urban heat island, but also increase people’s thermal comfort. By the way, urban green areas could also induce a positive effect on the local urban air quality. But attention has to be paid to the assortment of the tree species. More or less all tree species are emitting biogenic volatile organic compounds in different concentration. These serve as precursors for the formation of ozone near the ground. So near surface ozone has the ability to react with different particulate matters and could become toxic, due to oxidation or nitrification. This causes inflammations and inspired allergens may increase the risk of a respiratory disease. Therefore, an analysis and assessment of the urban green area air quality could help to make a statement about the recreational effect of these areas in dependence of the leading vegetation and for that matter for the exposure to ozone. By the help of these the results can be used as a guidance of urban planning taking into account the influence of biogenic emission as a function of actual weather conditions.

The Kinetic Study on the Anionic Polymerization of Isoprene

The kinetic study of the anionic polymerization of isoprene is carried out in tetrahy-drofuran(THF), using n-BuLi as initiator. Kinetic parameters are obtained, which comprise chain propagation rate constant, kp, and partial rate constants, k3 and k4 + k5, propagation orders with respect to monomer and active species concentrations, α and β, real activation energy, E, as well as partial activation energies, E3 and E4+5 and so on. The relationship between the microstruc-ture of polyisoprene and the ratio of [THF]/[n-BuLi] has been investigated. On the basis of the studies mentioned above, a reasonable mechanism of the anionic polymerization of isoprene in THF is proposed.

Chemical composition and size distribution of secondary organic aerosol formed from the photooxidation of isoprene

Photooxidation of isoprene leads to the formation of secondary organic aerosol （SOA）. In this study, the chemical composition of SOA formed from OH-initiated photooxidation of isoprene has been investigated with gas chromatography/mass spectrometry （GC/MS） and a home-made aerosol time-of-fiight mass spectrometer. Sampling particles generated in a home-made smog chamber. The size distribution of SOA particles was detected by a TSI 3321 aerodynamic particle size spectrometer in real time. Results showed that SOA created by isoprene photooxidation was predominantly in the form of fine particles, which have diameters less than 2.5 μm. The obtained mass spectra of individual particles show that products of the OH-initiated oxidation of isoprene contain methyl vinyl ketone, methacrolein, formaldehyde, and some other hydroxycarbonyls. The possible reaction mechanisms leading to these products were also discussed.

Synthesis of Poly(styrene-b-isoprene-b-styrene)viaNitroxide-mediated Radical Polymerization by a Novel Alkoxyamine

Bifunctional alkoxyamine bis-TIPNO derived from 2,2,5-trimethyl-4-phenyl-3-azahexane-3-oxyl （TIPNO） and α, ω-alkyl bromide by atom transfer radical addition（ATRA） was employed as “biradical initiator” for nitroxide-mediated radical polymerization（NMRP） of isoprene and styrene. The kinetics study for the polymerization of styrene at different time showed living features. The poly（styrene-b-isoprene-b-styrene） （SIS） copolymers have two glass transition temperatures, indicating the immiscibility of the corresponding blocks.

Synthesis and Characterization of Poly(isoprene-b-butyl methacrylate) Block Copolymer in the Presence of Rare Earth Catalyst

Poly(isoprene-b-butyl methacrylate) block copolymer with a high molecular weight was synthesized in the presence of rare earth coordination catalyst [RE(P 204 ) 3-Al( i Bu) 3 DBE]. The block copolymer was characterized by means of GPC,IR,DSC,NMR and elemental analysis. The block copolymer prepared has two glass transition temperatures: -75 6 ℃ and 32 2 ℃. The microstructure of the diblock copolymer possessed 96 8% cis 1,4 addition for the isoprene segment and a 70% syndiotacticity for the butyl methacrylate segment.

THE REGIOSELECTIVE SYNTHESIS OF NOVEL PHENYLSULFONES BEARING ISOPRENE UNITS

The facile and regioselective synthesis of some novel phenylsulfones bearing isoprene units were accomplished by using the corresponding terpenoid compounds as the starting material through ene-chlorination and phenylsulfonyl group substritution steps. The yields are excellent and the experimental conditions are very mild.

Copolymerization of Butadiene and Isoprene by NdC_6H_6(AlCl_4)_3-AlR_3 System

This paper reported the copolymerization results of butadiene and isoprene catalyzed by NdC_6H_6(AlCl_4)_3-AlR_3 system.The results show that the catalytic activity is dependent on the weight ratio of butadiene and isoprene,the concentration of comonomers,the nature of alkylaluminium and the polymerization temperature as well.High contents of 1,4 microstructure of copolymer are obtained by the novel catalyst system.

Determination of breath isoprene in 109 suspected lung cancer patients using cavity ringdown spectroscopy

Background:Lung cancer is one of the most common malignant tumors worldwide.Currently,effective screening methods for early lung cancer are still scarce.Breath analysis provides a promising method for the pre-screening or early screening of lung cancer.Isoprene is a potential and important breath biomarker of lung cancer.Material and Methods:To investigate the clinical value of isoprene for diagnosing lung cancer patients,a cavity ringdown spectroscopy(CRDS)based near-real time,sensitive analysis method of breath isoprene is developed in our lab.In this paper,92 breath samples from lung cancer patients,17 breath samples from patients with benign lesions,and 107 breath samples from healthy people were collected.Results:Research indicates that breath isoprene concentration is significantly higher in healthy individuals(221:3±122:2 ppbv)than in patients with lung cancer(112:0±36:6 ppbv)and benign lung lesions(127:9±41:2 ppbv).The result of Receiver Operating Characteristic(ROC)curve suggests that the concentration of isoprene is meaningful for the diagnosis of lung cancer(AUC=0:822,sensitivity=63:6%,specificity=90:2%,P<0:01).Conclusion:This study demonstrates that the CRDS breath isoprene analysis system can effectively analyze a large sample of human breath isoprene,and preliminarily confirms the use of breath isoprene as a biomarker for lung diseases.

Renewable biosynthesis of isoprene from wastewater through a synthetic biology approach:the role of individual organic compounds

The biosynthesis of isoprene offers a more sustainable alternative to fossil fuel-based approaches,yet its success has been largely limited to pure organic compounds and the cost remains a challenge.This study proposes a waste-to-wealth strategy for isoprene biosynthesis utilizing genetically engineered E.coli bacteria to convert organic waste from real food wastewater.The impact of organic compounds present in wastewater on E.coli growth and isoprene production was systematically investigated.The results demonstrated that with filtration pretreatment of wastewater,isoprene yield,and production achieved 115 mg/g COD and 7.1 mg/(L·h),respectively.Moreover,even without pretreatment,isoprene yield only decreased by~24%,indicating promising scalability.Glucose,maltose,glycerol,and lactate are effective substrates for isoprene biosynthesis,whereas starch,protein,and acetate do not support E.coli growth.The optimum C/N ratio for isoprene production was found to be 8:1.Furthermore,augmenting essential nutrients in wastewater elevated the isoprene yield increased to 159 mg/g COD.The wastewater biosynthesis significantly reduced the cost(44%–53%decrease,p-value<0.01)and CO_(2)emission(46%–55%decrease,p-value<0.01)compared with both sugar fermentation and fossil fuel–based refining.This study introduced a more sustainable and economically viable approach to isoprene synthesis,offering an avenue for resource recovery from wastewater.

Impacts of global biogenic isoprene emissions on surface ozone during 2000-2019

Biogenic isoprene is an important precursor of tropospheric ozone(O_(3)).Here,a coupled chemistry-vegetation model was used to quantify the contributions of isoprene emissions to surface O_(3)pollution on the global scale during 2000-2019.The biogenic isoprene emissions showed high values in mid-low latitudes and seasonal peaks in the summer hemispheres.They promote global surface O_(3)concentrations by 1.75 ppbv annually with regional hotspots of 4.39 ppbv(8.8%)in China and 5.36 ppbv(11.1%)in the U.S.in boreal summer.In the past two decades,isoprene emissions increased by 1.32 TgC yr^(−1)(0.67%yr^(−1))in the Northern Hemisphere but decreased by 0.71 TgC yr^(−1)(0.44%yr^(−1))in the Southern Hemisphere.Such changes of isoprene made opposite contributions to the surface O_(3)trend,with 0.26 ppbv yr^(−1)in eastern China but−0.32 ppbv yr^(−1)in the southeastern U.S.due to the changes in the background regime of chemical reactions.The impact of anthropogenic changes on the O_(3)trend is consistent with that of biogenic isoprene,but two to four times stronger in magnitude.This study revealed that the effective control of anthropogenic NO_(x)emissions could mitigate regional O_(3)pollution even with the increased isoprene emissions under global warming.

Manganese porphyrin-mediated aerobic epoxidation of propylene with isoprene: A new strategy for simultaneously preparing propylene epoxide and isoprene monoxide

The direct epoxidation of propylene by O_(2) is a significant and challenging topic. The key factor for this homogeneous aerobic epoxidation is the activation of molecular oxygen under mild conditions. In this work,the aerobic epoxidation of propylene catalyzed by manganese porphyrins was achieved in the presence of isoprene. Isoprene contains an allyl methyl group, and the α-H can be easily removed to achieve the activation of molecular oxygen. The conversion of propylene was 38% and the selectivity toward propylene oxide(PO) was up to 87%. The role of isoprene was demonstrated, and a plausible mechanism was proposed. The protocol reported herein is expected to provide a strategy for the simultaneous preparation of propylene oxide and isoprene monoxide.

Heterologous Expression of the Mevalonic Acid Pathway in Cyanobacteria Enhances Endogenous Carbon Partitioning to Isoprene

Heterologous expression of the isoprene synthase gene in the cyanobacterium Synechocystis PCC 6803 conferred upon these microorganisms the property of photosynthetic isoprene （C5H8） hYdrocarbons production. Continuous production of isoprene from CO2 and H2O was achieved in the light, occurring via the endogenous methylerythritolphosphate （MEP） pathway, in tandem with the growth of Synechocystis. This work addressed the issue of photosynthetic carbon partitioning between isoprene and biomass in Synechocystis. Evidence is presented to show heterologous genomic integration and cellular expression of the mevalonic acid （MVA） pathway genes in Synechocystis endowing a non-native pathway for carbon flux amplification to isopentenyl-diphosphate （IPP） and dimethylallyl-diphosphate （DMAPP） precursors of isoprene. Heterologous expression of the isoprene synthase in combination with the MVA pathway enzymes resulted in photosynthetic isoprene yield improvement by approximately 2.5-fold, compared with that measured in cyanobacteria transformed with the isoprene synthase gene only. These results suggest that the MVA pathway introduces a bypass in the flux of endogenous cellular substrate in Synechocystis to IPP and DMAPP, overcoming flux limitations of the native MEP pathway. The work employed a novel chromosomal integration and expression of synthetic gene operons in Synechocystis, comprising up to four genes under the control of a single promoter, and expressing three operons simultaneously. This is the first time an entire biosynthetic pathway with seven recombinant enzymes has been heterologously expressed in a photosynthetic microorganism. It constitutes contribution to the genetic engineering toolkit of photosynthetic microorganisms and a paradigm in the pursuit of photosynthetic approaches for the renewable generation of high-impact products.