Highly Oxygenated Molecules from Atmospheric Autoxidation of Hydrocarbons : A Prominent Challenge for Chemical Kinetics Studies

Recent advances in chemical ionization mass spectrometry have allowed the detection of a new group of compounds termed highly oxygenated molecules (HOM). These are atmospheric oxidation products of volatile organic compounds (VOC) retaining most of their carbon backbone, and with O/C ratios around unity. Owing to their surprisingly high yields and low vapor pressures, the importance of HOM for aerosol formation has been easy to verify. However, the opposite can be said concerning the exact formation pathways of HOM from major aerosol precursor VOC. While the role of peroxy radical autoxidation, i.e., consecutive intramolecular H-shifts followed by O-2 addition, has been recognized, the detailed formation mechanisms remain highly uncertain. A primary reason is that the autoxidation process occurs on sub-second timescales and is extremely sensitive to environmental conditions like gas composition, temperature, and pressure. This, in turn, poses a great challenge for chemical kinetics studies to be able to mimic the relevant atmospheric reaction pathways, while simultaneously using conditions suitable for studying the short-lived radical intermediates. In this perspective, we define six specific challenges for this community to directly observe the initial steps of atmospherically relevant autoxidation reactions and thereby facilitate vital improvements in the understanding of VOC degradation and organic aerosol formation. (C) 2017 Wiley Periodicals, Inc.Peer reviewe

Secondary organic aerosol reduced by mixture of atmospheric vapours

Secondary organic aerosol contributes to the atmospheric particle burden with implications for air quality and climate. Biogenic volatile organic compounds such as terpenoids emitted from plants are important secondary organic aerosol precursors with isoprene dominating the emissions of biogenic volatile organic compounds globally. However, the particle mass from isoprene oxidation is generally modest compared to that of other terpenoids. Here we show that isoprene, carbon monoxide and methane can each suppress the instantaneous mass and the overall mass yield derived from monoterpenes in mixtures of atmospheric vapours. We find that isoprene 'scavenges' hydroxyl radicals, preventing their reaction with monoterpenes, and the resulting isoprene peroxy radicals scavenge highly oxygenated monoterpene products. These effects reduce the yield of low-volatility products that would otherwise form secondary organic aerosol. Global model calculations indicate that oxidant and product scavenging can operate effectively in the real atmosphere. Thus highly reactive compounds (such as isoprene) that produce a modest amount of aerosol are not necessarily net producers of secondary organic particle mass and their oxidation in mixtures of atmospheric vapours can suppress both particle number and mass of secondary organic aerosol. We suggest that formation mechanisms of secondary organic aerosol in the atmosphere need to be considered more realistically, accounting for mechanistic interactions between the products of oxidizing precursor molecules (as is recognized to be necessary when modelling ozone production).Peer reviewe

N-Glycan Modification in Aspergillus Species▿

The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional α-1,2-mannosidase producing increased amounts of N-glycans of the Man5GlcNAc2 type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan5GlcNac2 N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man3GlcNAc2 N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi

Characterization of the Pichia pastoris protein-O-mannosyltransferase gene family.

The methylotrophic yeast, Pichiapastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of β-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P. pastoris, thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P. pastoris is the transfer of mannose from dolichol-phosphomannose to a target protein in the yeast secretory pathway by members of the protein-O-mannosyltransferase (PMT) family. In this report we identify and characterize the members of the P. pastoris PMT family. Like Candida albicans, P. pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively). The remaining sub-family, PMT4, has only one member (PMT4). Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of O-mannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P. pastoris platform as a suitable system for the production of therapeutic glycoproteins

Western blot analysis of an IgG1 expressed in the presence of PMTi-3.

<p>The <i>PMT</i> wild type strain YGLY4280 was grown in 96 well deep-well plates and induced in medium containing no PMTi-3 (C) or increasing concentrations of PMTi-3 (0.0187, 0.0375, 0.075, 0.15, 0.3, 0.625, 1.25, 2.5, 5, 10 µg/ml). After overnight induction, 7 µl of each supernatant was separated by polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto nitrocellulose membranes. IgG1 was detected using an anti-human IgG (heavy and light chain) antibody. Hyperglycosylated heavy-chain (indicated by an asterisk) is barely detectable at PMTi-3 concentrations of 0.15 µg/ml and higher, however strain growth and fitness (strain lysis) was still unaffected at PMTi-3 concentrations of up to 1.25 µg/ml. A representation of the PMTi-3 increase in concentration and the relative reduction in cell fitness are illustrated by <i>light</i> and <i>dark gray triangles</i> respectively.</p

Overview of <i>PMT</i> knockout experiment.

<p>(A) The PCR overlap and <i>PMT</i> knockout procedure. First a linear gene deletion allele was constructed the following way: a region 5’ of the <i>PMT</i> ORF was amplified using primers 1 and 2, the selectable marker was amplified using primers 3 and 4, and a region 3’ of the <i>PMT</i> ORF was amplified using primers 5 and 6. The primers in parentheses were added to the schematic to illustrate that primers 2 and 3, as well as primers 4 and 5, are complementary to each other (thereby creating the overlaps). The amplified products were then used as templates together with primers 1 and 6 in the overlap reaction. The linear deletion allele was then integrated into the <i>P</i><i>. pastoris</i> genome via homologous recombination, and knockouts were selected on plates supplemented with the appropriate antibiotic. (B) Western blot analysis of Kringle 1-3 protein (K1-3) expressed in wild type and <i>PMT</i> knockout strains. Strains JC53 (wild type), JC55 (<i>Δpmt1</i>), JC66 (<i>Δpmt2</i>), JC65 (<i>Δpmt4</i>), and JC51 (<i>Δpmt5/6</i>) were grown and induced in shake flasks. Seven µl of each supernatant was separated by polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto nitrocellulose membranes. K1-3 protein was detected using an anti-His antibody. O-glycan site occupancy (Occ.) was determined as described in Materials and Methods. The expected mass of the fully deglycosylated protein is 44.2 kDa, and the band at 36 kDa probably represents proteolytic cleavage.</p

Inhibitor sensitivity of <i>PMT</i> knockouts.

<p>For dilution assays, yeast cells were grown overnight in YSD and then adjusted to an OD600 of 1.0 using YSD, diluted in 10-fold increments, and then 3 µl of each dilution were spotted onto solid YSD media with varying concentrations of PMTi-3. The picture of the plate without PMTi-3 was taken after 48 hours, and the picture of the plates containing PMTi-3 were taken after 96 hours of incubation at 25^°C. YGLY4140 is the parent strain of YGLY6001 and YGLY4329, YGLY733 is the parent strain of YGLY3773 and YGLY5968, and NRRL -Y11430 is the parent strain of YGLY35032.</p

<i>PMT</i> expression profile by microarray analysis.

<p>The relative levels of PMT mRNA levels were assessed by microarray analysis under glycerol (<i>shaded bars</i>) or methanol (<i>black bars</i>) growth conditions as described in the Materials and Methods Section. mRNA levels for <i>ACT1</i> and <i>GPD</i> have been added for reference.</p