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cis , cis -Muconic acid for downstream separation and catalytic upgrading to adipic acid for nylon-6,6 polymerization.
Muconic acid is an important platform molecule for production of bio-based polymers. To date, cis,cis-muconic acid (ccMA) has been produced from multiple types of carbon sources including carbohydrates and aromatic compounds via cultivation with genetically engineered microbes. Accurate analysis of muconic acid has previously been a challenge due to ccMA isomerization into cis,trans-muconic acid (ctMA). This Laboratory Analytical Procedure (LAP) is used to separately quantify ccMA and ctMA as the total sum of muconic acid present in the liquid fraction of pretreated biological samples.
Cis isomers of carotenoids play important roles in light harvesting and photoprotection in photosynthetic bacteria, such as the reaction center in purple bacteria and the photosynthetic apparatus in cyanobacteria. Carotenoids containing carbonyl groups are involved in efficient energy transfer to chlorophyll in light-harvesting complexes, and their intramolecular charge–transfer (ICT) excited states are known to be important for this process. Previous studies, using ultrafast laser spectroscopy, have focused on the central-cis isomer of carbonyl-containing carotenoids, revealing that the ICT excited state is stabilized in polar environments. However, the relationship between the cis isomer structure and the ICT excited state has remained unresolved. In this study, we performed steady-state absorption and femtosecond time-resolved absorption spectroscopy on nine geometric isomers (7-cis, 9-cis, 13-cis, 15-cis, 13′-cis, 9,13′-cis, 9,13-cis, 13,13′-cis, and all-trans) of β-apo-8′-carotenal, whose structures are well-defined, and discovered correlations between the decay rate constant of the S1 excited state and the S0−S1 energy gap, as well as between the position of the cis-bend and the degree of stabilization of the ICT excited state. Our results demonstrate that the ICT excited state is stabilized in polar environments in cis isomers of carbonyl-containing carotenoids and suggest that the position of the cis-bend plays an important role in the stabilization of the excited state.
Abstract not provided
HOCO is an important intermediate in combustion and atmospheric processes because the OH + CO → H + CO{sub 2} reaction represents the final step for the production of CO{sub 2} in hydrocarbon oxidation, and theoretical studies predict that this reaction proceeds via various intermediates, the most important being this radical. Isotopic investigations of trans- and cis-HOCO have been undertaken using Fourier transform microwave spectroscopy and millimeter-wave double resonance techniques in combination with a supersonic molecular beam discharge source to better understand the formation, chemical bonding, and molecular structures of this radical pair. We find that trans-HOCO can be produced almost equally well from either OH + CO or H + CO{sub 2} in our discharge source, but cis-HOCO appears to be roughly two times more abundant when starting from H + CO{sub 2}. Using isotopically labelled precursors, the OH + C{sup 18}O reaction predominately yields HOC{sup 18}O for both isomers, but H{sup 18}OCO is observed as well, typically at the level of 10%-20% that of HOC{sup 18}O; the opposite propensity is found for the {sup 18}OH + CO reaction. DO + C{sup 18}O yields similar ratios between DOC{sup 18}O and D{sup 18}OCO as those found for OH + C{sup 18}O, suggesting that some fraction of HOCO (or DOCO) may be formed from the back-reaction H + CO{sub 2}, which, at the high pressure of our gas expansion, can readily occur. The large {sup 13}C Fermi-contact term (a{sub F}) for trans- and cis-HO{sup 13}CO implicates significant unpaired electronic density in a σ-type orbital at the carbon atom, in good agreement with theoretical predictions. By correcting the experimental rotational constants for zero-point vibration motion calculated theoretically using second-order vibrational perturbation theory, precise geometrical structures have been derived for both isomers.
Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean ( Phaseolus coccineus ) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we use site-directed mutagenesis experiments in transgenic tobacco globularstage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis -regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. Lastly, a homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.
Pure samples of cis,cis- and trans,trans-1,4-difluorobutadiene-2-d1 have been synthesized, and high-resolution (0.0015 cm-1) infrared spectra have been recorded for these nonpolar molecules in the gas phase. For the cis,cis isomer, the rotational structure in two C-type bands at 775 and 666 cm-1 and one A-type band at 866 cm-1 has been analyzed to yield a combined set of 2020 ground state combination differences (GSCDs). Ground state rotational constants fit to these GSCDs are A0 = 0.4195790(4), B0 = 0.0536508(8), and C0 = 0.0475802(9) cm-1. For the trans,trans isomer, three Ctype bands at 856, 839, and 709 cm-1 have been investigated to give a combined set of 1624 GSCDs. Resulting ground state rotational constants for this isomer are A0 = 0.9390117(8), B0 = 0.0389225(4), and C0 = 0.0373778(3) cm-1. Small inertial defects confirm the planarity of both isomers in the ground state. Upper state rotational constants have been determined for most of the transitions. The ground state rotational constants for the two isotopologues will contribute to the data set needed for determining semiexperimental equilibrium structures for the nonpolar isomers of 1,4- difluorobutadiene.
The nature of reactive intermediates and the mechanism of the cis-dihydroxylation of arenes and olefins by Rieske dioxygenases and synthetic nonheme iron catalysts have been the topic of intense research over the past several decades. In this study, we report that a spectroscopically well characterized mononuclear nonheme iron(III)-peroxo complex reacts with olefins and naphthalene derivatives, yielding iron(III) cycloadducts that are isolated and characterized structurally and spectroscopically. Further, kinetics and product analysis reveal that the nonheme iron(III)-peroxo complex is a nucleophile that reacts with olefins and naphthalenes to yield cis-diol products. The present study reports the first example of the cis-dihydroxylation of substrates by a nonheme iron(III)-peroxo complex that yields cis-diol products.
ABSTRACT The platform chemical cis,cis- muconic acid ( cc MA) provides facile access to a number of monomers used in the synthesis of commercial plastics. It is also a metabolic intermediate in the β-ketoadipic acid pathway of many bacteria and, therefore, a current target for microbial production from abundant renewable resources via metabolic engineering. This study investigates Novosphingobium aromaticivorans DSM12444 as a chassis for the production of cc MA from biomass aromatics. The N. aromaticivorans genome predicts that it encodes a previously uncharacterized protocatechuic acid (PCA) decarboxylase and a catechol 1,2-dioxygenase, which would be necessary for the conversion of aromatic metabolic intermediates to cc MA. This study confirmed the activity of these two enzymes in vitro and compared their activity to ones that have been previously characterized and used in cc MA production. From these results, we generated one strain that is completely derived from native genes and a second that contains genes previously used in microbial engineering synthesis of this compound. Both of these strains exhibited stoichiometric production of cc MA from PCA and produced greater than 100% yield of cc MA from the aromatic monomers that were identified in liquor derived from alkaline pretreated biomass. Our results show that a strain completely derived from native genes and one containing homologs from other hosts are both capable of stoichiometric production of cc MA from biomass aromatics. Overall, this work combines previously unknown aspects of aromatic metabolism in N. aromaticivorans and the genetic tractability of this organism to generate strains that produce cc MA from deconstructed biomass. IMPORTANCE The production of commodity chemicals from renewable resources is an important goal toward increasing the environmental and economic sustainability of industrial processes. The aromatics in plant biomass are an underutilized and abundant renewable resource for the production of valuable chemicals. However, due to the chemical composition of plant biomass, many deconstruction methods generate a heterogeneous mixture of aromatics, thus making it difficult to extract valuable chemicals using current methods. Therefore, recent efforts have focused on harnessing the pathways of microorganisms to convert a diverse set of aromatics into a single product. Novosphingobium aromaticivorans DSM12444 has the native ability to metabolize a wide range of aromatics and, thus, is a potential chassis for conversion of these abundant compounds to commodity chemicals. This study reports on new features of N. aromaticivorans that can be used to produce the commodity chemical cis,cis -muconic acid from renewable and abundant biomass aromatics.
Abstract Corynebacterium glutamicum ATCC 13032 is a promising microbial chassis for industrial production of valuable compounds, including aromatic amino acids derived from the shikimate pathway. In this work, we developed two whole-cell, transcription factor based fluorescent biosensors to track cis,cis-muconic acid (ccMA) and chorismate in C. glutamicum. Chorismate is a key intermediate in the shikimate pathway from which value-added chemicals can be produced, and a shunt from the shikimate pathway can divert carbon to ccMA, a high value chemical. We transferred a ccMA-inducible transcription factor, CatM, from Acinetobacter baylyi ADP1 into C. glutamicum and screened a promoter library to isolate variants with high sensitivity and dynamic range to ccMA by providing benzoate, which is converted to ccMA intracellularly. The biosensor also detected exogenously supplied ccMA, suggesting the presence of a putative ccMA transporter in C. glutamicum, though the external ccMA concentration threshold to elicit a response was 100-fold higher than the concentration of benzoate required to do so through intracellular ccMA production. We then developed a chorismate biosensor, in which a chorismate inducible promoter regulated by natively expressed QsuR was optimized to exhibit a dose-dependent response to exogenously supplemented quinate (a chorismate precursor). A chorismate–pyruvate lyase encoding gene, ubiC, was introduced into C. glutamicum to lower the intracellular chorismate pool, which resulted in loss of dose dependence to quinate. Further, a knockout strain that blocked the conversion of quinate to chorismate also resulted in absence of dose dependence to quinate, validating that the chorismate biosensor is specific to intracellular chorismate pool. The ccMA and chorismate biosensors were dually inserted into C. glutamicum to simultaneously detect intracellularly produced chorismate and ccMA. Biosensors, such as those developed in this study, can be applied in C. glutamicum for multiplex sensing to expedite pathway design and optimization through metabolic engineering in this promising chassis organism. One-Sentence Summary High-throughput screening of promoter libraries in Corynebacterium glutamicum to establish transcription factor based biosensors for key metabolic intermediates in shikimate and β-ketoadipate pathways.
Highlights: • Engineered C. glutamicum produced CCM, valued for plastic and polymer production. • The CCM yield from glucose in the optimized strain was about 22% (mol/mol). • CCM productivity was enhanced by redirecting carbon flux in non-PTS C. glutamicum. Cis,cis-muconic acid (CCM) is a biochemical material that can be used for the production of various plastics and polymers and is particularly gaining attention as an adipic acid precursor for the synthesis of nylon-6,6. In the current study, the production of CCM was first attempted by introducing a newly developed protocatechuate (PCA) decarboxylase from Corynebacterium glutamicum 13032 to inha103, which completed the biosynthetic pathway therein. To improve CCM productivity, a phosphoenol pyruvate (PEP)-dependent phosphotransferase system (PTS) that consumed the existing glucose was developed, in the form of a strain with a non-PTS that did not consume PEP. To improve glucose uptake, we developed P25 strain, in which iolR (a transcriptional regulator gene) was additionally deleted. Strain P28, a P25 derivative expressing PCA decarboxylase, produced 4.01 g/L of CCM, which was 14% more than that produced by the parental strain. Moreover, strains P29 and P30, with an active pentose phosphate pathway and overexpressing important genes (qsuB) in the metabolic pathway, produced 4.36 and 4.5 g/L of CCM, respectively. Particularly, the yield per glucose in strain P30 was similar to that of the fed-batch culture of Escherichia coli, which has the highest reported yield of 22% (mol/mol). These results are underpinned by the characteristics of the non-PTS with increased PEP availability and a strain with deletion of the iolR gene, which greatly increased glucose uptake.
Samples of cis,cis- and trans,trans-1,4-difluorobutadiene-1- d1 and of trans,trans-1,4-difluorobutadiene-1,4-d2 have been synthesized, and high-resolution (≤0.0018 cm-1) infrared spectra of these substances have been recorded in the gas phase. Analysis of the rotational structure, mostly in C-type bands, has yielded ground state rotational constants. For the two 1-d1 species more than one band has been analyzed. For the 1,4-d2 species only one band was available for analysis. However, good agreement between the experimental centrifugal distortion constants and those predicted with a B3LYP/cc-pVTZ model give strong support to the analysis of the very dense spectrum. The ground state rotational constants are a contribution to finding semiexperimental equilibrium structures of the two nonpolar isomers of 1,4- difluorobutadiene.
One-electron oxidation of the μ-oxo dimer (cis,cis-[Ru III (bpy) 2 (OH 2 )] 2 O 4+ , {3,3} ) to {3,4} by S 2 O 8 2- can be described by three concurrent reaction pathways corresponding to the three protic forms of {3,3} . Free energy correlations of the rate constants, transient species dynamics determined by pulse radiolysis, and medium and temperature dependencies of the alkaline pathway all suggest that the rate determining step in these reactions is a strongly non-adiabatic dissociative electron transfer within a precursor ion pair leading to the {3,4} |SO 4 2- |SO 4 •- ion triple. As deduced from the SO 4 •- scavenging experiments with 2-propanol, the SO 4 •- radical then either oxidizes {3,4} to {4,4} within the ion triple, effecting a net two-electron oxidation of {3,3} , or escapes in solution with ~25 % probability to react with additional {3,3} and {3,4} , that is, effecting sequential one-electron oxidations. The reaction model presented also invokes rapid {3,3} + {4,4} → 2 {3,4} comproportionation, for which k com ~5×10 7 M -1 s -1 was independently measured. The model provides an explanation for the observation that despite favorable energetics, no oxidation beyond the {3,4} state was detected. As a result, the indiscriminate nature of oxidation by SO 4 •- indicates that its fate must be quantitatively determined when using S 2 O 8 2- as an oxidant
Unlocking the isomerization of cis , cis -muconic acid to Diels–Alder active trans , trans -muconic acid for the production of renewable cyclic diacid monomers.
Conjugated diene molecules are highly reactive upon photoexcitation and can relax through multiple reaction channels that depend on the position of the double bonds and the degree of molecular rigidity. Understanding the photoinduced dynamics of these molecules is crucial for establishing general rules governing the relaxation and product formation. Here, in this study, we investigate the femtosecond time-resolved photoinduced excited-state structural dynamics of cis,cis-1,3-cyclooctadiene, a large-flexible cyclic conjugated diene molecule, upon excitation with 200 nm using mega-electron-volt ultrafast electron diffraction and trajectory surface hopping dynamics simulations. We tracked the photoinduced structural changes from the Franck–Condon region through the conical intersection seam to the ground state. Our findings revealed a novel primary reaction coordinate involving ring distortion, where the ring stretches along one axis and compresses along the perpendicular axis. The nuclear wavepacket remains compact along this reaction coordinate until it reaches the conical intersection seam, and it rapidly spreads as it approaches the ground state, where multiple products are formed.
We report novel experimental strategies that should prove instrumental in extending the vibrational and rotational assignments of the S{sub 1} state of acetylene, C{sub 2}H{sub 2}, in the region of the cis-trans isomerization barrier. At present, the assignments are essentially complete up to ∼500 cm{sup −1} below the barrier. Two difficulties arise when the assignments are continued to higher energies. One is that predissociation into C{sub 2}H + H sets in roughly 1100 cm{sup −1} below the barrier; the resulting quenching of laser-induced fluorescence (LIF) reduces its value for recording spectra in this region. The other difficulty is that tunneling through the barrier causes a staggering in the K-rotational structure of isomerizing vibrational levels. The assignment of these levels requires data for K values up to at least 3. Given the rotational selection rule K′ − ℓ{sup ′′} = ± 1, such data must be obtained via excited vibrational levels of the ground state with ℓ{sup ′′} > 0. In this paper, high resolution H-atom resonance-enhanced multiphoton ionization spectra are demonstrated to contain predissociated bands which are almost invisible in LIF spectra, while preliminary data using a hyperthermal pulsed nozzle show that ℓ{sup ′′} = 2 states can be selectively populated in a jet, giving access to K′ = 3 states in IR-UV double resonance.
A high level ab initio calculation of the cis-trans isomerization barrier height in the first excited singlet electronic state of acetylene is found to agree very well with a recent experimental determination.
We have performed trajectory surface hopping dynamics for cis,cis-1,3-cyclooctadiene to investigate the photochemical pathways involved after being excited to the S 1 state. Our calculations reveal ultrafast decay to the ground state, facilitated by conical intersections involving distortions around the double bonds. The main distortions are localized on one double bond, involving twisting and pyramidalization of one of the carbons of that double bond (similar to ethylene), while a limited number of trajectories decay via delocalized (non-local) twisting of both double bonds. The interplay between local and non-local distortions is important in our understanding of photoisomerization in conjugated systems. The calculations show that a broad range of the conical intersection seam space is accessed during the non-adiabatic events. Several products formed on the ground state have also been observed.