•Protocatechuate decarboxylation is a rate-limiting step in muconate production.•We identified a way to enhance protocatechuate decarboxylase activity.•Muconate production from vanillin can be ...achieved via the enhanced decarboxylase.•Muconate may cause a feedback inhibition on vanillate demethylase in the pathway.
The decarboxylation reaction of protocatechuate has been described as a bottleneck and a rate-limiting step in cis,cis-muconate (ccMA) bioproduction from renewable feedstocks such as sugar. Because sugars are already in high demand in the development of many bio-based products, our work focuses on improving protocatechuate decarboxylase (Pdc) activity and ccMA production in particular, from lignin-related aromatic compounds. We previously had transformed an Escherichia coli strain using aroY, which had been used as a protocatechuate decarboxylase encoding gene from Klebsiella pneumoniae subsp. pneumoniae A170-40, and inserted other required genes from Pseudomonas putida KT2440, to allow the production of ccMA from vanillin. This recombinant strain produced ccMA from vanillin, however the Pdc reaction step remained a bottleneck during incubation. In the current study, we identify a way to increase protocatechuate decarboxylase activity in E. coli through enzyme production involving both aroY and kpdB; the latter which encodes for the B subunit of 4-hydroxybenzoate decarboxylase. This permits expression of Pdc activity at a level approximately 14-fold greater than the strain with aroY only. The expression level of AroY increased, apparently as a function of the co-expression of AroY and KpdB. Our results also imply that ccMA may inhibit vanillate demethylation, a reaction step that is rate limiting for efficient ccMA production from lignin-related aromatic compounds, so even though ccMA production may be enhanced, other challenges to overcome vanilate demethylation inhibition still remain.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Brown rot (BR) decay mechanisms employ carbohydrate-active enzymes (CAZymes) as well as a unique non-enzymatic chelator-mediated Fenton (CMF) chemistry to deconstruct lignocellulosic materials. ...Unlike white rot fungi, BR fungi lack peroxidases for lignin deconstruction, and also lack some endoglucanase/cellobiohydrolase activities. The role that the CMF mechanism plays in "opening up" the wood cell wall structure in advance of enzymatic action, and any interaction between CMF constituents and the selective CAZyme suite that BRs possess, is still unclear. Expression patterns for CMF redox metabolites and lytic polysaccharide monooxygenase (LPMO-AA9 family) genes showed that some LPMO isozymes were upregulated with genes associated with CMF at early stages of brown rot by
. In the structural studies, wood decayed by the
was compared to CMF-treated wood, or CMF-treated wood followed by treatment with either the early-upregulated LPMO or a commercial CAZyme cocktail. Structural modification of decayed/treated wood was characterized using small angle neutron scattering. CMF treatment produced neutron scattering patterns similar to that of the BR decay indicating that both systems enlarged the nanopore structure of wood cell walls to permit enzyme access. Enzymatic deconstruction of cellulose or lignin in raw wood samples was not achieved via CAZyme cocktail or LPMO enzyme action alone. CMF treatment resulted in depolymerization of crystalline cellulose as attack progressed from the outer regions of individual crystallites. Multiple pulses of CMF treatment on raw wood showed a progressive increase in the spacing between the cellulose elementary fibrils (EFs), indicating the CMF eroded the matrix outside the EF bundles, leading to less tightly packed EFs. Peracetic acid delignification treatment enhanced subsequent CMF treatment effects, and allowed both enzyme systems to further increase spacing of the EFs. Moreover, even after a single pulse of CMF treatment, both enzymes were apparently able to penetrate the cell wall to further increase EF spacing. The data suggest the potential for the early-upregulated LPMO enzyme to work in association with CMF chemistry, suggesting that
may have adopted mechanisms to integrate non-enzymatic and enzymatic chemistries together during early stages of brown rot decay.
As one of the most abundant materials in nature, lignin has been used widely in co-generation operations and for fine chemicals and bio-fuels production. These uses, although important, are of ...relatively low value. Lignin contains many aromatic compounds with useful structures, and it is potentially more profitable to produce high-value fine chemicals from the low-molecular weight lignin fraction while using the high-molecular weight fraction for fuel or other applications. A transgenic P. putida bacterial strain PDHV85 was developed with the capability to convert vanillin, vanillic acid, and syringaldehyde to 2-pyrone-4,6-dicarboxylic acid (PDC), a novel platform chemical that can produce a variety of bio-based polymers. Initial testing with vanillin showed promise for lignin conversion. Testing for this, we used kraft lignin, Japanese cedar (Cryptomeria japonica), or birch (Betula platyphylla) to represent some of the most abundant industrial lignin sources from softwood and hardwood. Repeated manipulation of culture conditions and strain adaptation allowed conversion of these extracts to PDC by PDHV85, which has not previously been reported in a bacterial strain. No inhibition was observed at 0.14 mg/mL kraft lignin extract, 1.14 mg/mL Japanese cedar extract, nor 1.15 mg/mL birch extract when using the optimized growth conditions.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
This study aims to clarify the role of variegatic acid (VA) in fungal attack by
Serpula lacrymans
, and also the generation and scavenging of reactive oxygen species (ROS) by the fungus. VA promotes ...a mediated Fenton reaction to generated ROS after oxalate solubilizes oxidized forms of iron. The fungal extracellular matrix (ECM) β-glucan scavenged ROS, and we propose this as a mechanism to protect the fungal hyphae while ROS generation is promoted to deconstruct the lignocellulose cell wall. A relatively high pH (4.4) also favored Fe(III) transfer from oxalate to VA as opposed to a lower pH (2.2) conditions, suggesting a pH-dependent Fe(III) transfer to VA employed by
S. lacrymans.
This permits ROS generation within the higher pH of the cell wall, while limiting ROS production near the fungal hyphae, while β-glucan from the fungal ECM scavenges ROS in the more acidic environments surrounding the fungal hyphae.
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CEKLJ, DOBA, FZAB, GEOZS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Eutypa dieback and Esca complex are fungal diseases of grape that cause large economic losses in vineyards. These diseases require, or are enhanced by, fungal consortia growth which leads to the ...deterioration of the wood tissue in the grapevine trunk; however, pathogenesis and the underlying mechanisms involved in the woody tissue degradation are not understood. We examined the role that the consortia fungal metabolome have in generating oxygen radicals that could potentially play a role in trunk decay and pathogenesis. Unique metabolites were isolated from the consortia fungi with some metabolites preferentially reducing iron whereas others were involved in redox cycling to generate hydrogen peroxide. Metabolite suites with different functions were produced when fungi were grown separately vs. when grown in consortia. Chelator-mediated Fenton (CMF) chemistry promoted by metabolites from these fungi allowed for the generation of highly reactive hydroxyl radicals. We hypothesize that this mechanism may be involved in pathogenicity in grapevine tissue as a causal mechanism associated with trunk wood deterioration/necrosis in these two diseases of grape.
• Premise of the study: Coniferous bordered pits are some of the most unique and fascinating microstructures of the lignified cell wall. The pit membrane consists of a margo and a torus region, hence ...facilitating both xylary water transport and also limiting air intrusion by pit aspiration. Additionally, bordered pits have been reported to play a decisive role in the control of rapid liquid flow via the shrinkage and swelling of pectin. The study of the nanostructural chemical composition of pit membranes has been difficult with common imaging/chemical techniques, which involve drying and/or coating of the samples.• Methods: Using fluorescent tagging and antibodies specific to pectin, and a His-tagged cellulose-binding module that reacts with crystalline cellulose, in combination with confocal laser scanning microscopy (CLSM) and 4Pi microscopy, we generated three-dimensional images of intact pit membranes.• Key results: With enhanced resolution in the z-direction of the 4Pi microscope, it was possible to distinguish cellulose in the torus and the margo strands of Pinus strobus. The torus was surrounded by pectin, and a pectin ring was found at the margin of the torus. We also found differences in the structure of the pit membrane between aspirated and unaspirated pits, with a displacement of pectin to form a ring-like structure, the collapse of a void in the interior of the torus, and an apparent change in the chemical structure of cellulosic components, during the aspiration process.• Conclusions: The 4Pi microscope is well suited to scanning pit membranes to discover previously undescribed anatomical features in bordered pits and can provide information on chemical composition when used in combination with appropriate probes.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NMLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
This work reviews the brown-rot fungal biochemical mechanism involved in the biodegradation of lignified plant cell walls. This mechanism has been acquired as an apparent alternative to the ...energetically expensive apparatus of lignocellulose breakdown employed by white-rot fungi. The mechanism relies, at least in the incipient stage of decay, on the oxidative cleavage of glycosidic bonds in cellulose and hemicellulose and the oxidative modification and arrangement of lignin upon attack by highly destructive oxygen reactive species such as the hydroxyl radical generated non-enzymatically via Fenton chemistry
. Modifications in the lignocellulose macrocomponents associated with this non-enzymatic attack are believed to aid in the selective, near-complete removal of polysaccharides by an incomplete cellulase suite and without causing substantial lignin removal. Utilization of this process could provide the key to making the production of biofuel and renewable chemicals from lignocellulose biomass more cost-effective and energy efficient. This review highlights the unique features of the brown-rot fungal non-enzymatic, mediated Fenton reaction mechanism, the modifications to the major plant cell wall macrocomponents, and the implications and opportunities for biomass processing for biofuels and chemicals.
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CEKLJ, DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Wood-decaying basidiomycetes are some of the most effective bioconverters of lignocellulose in nature, however the way they alter wood crystalline cellulose on a molecular level is still not well ...understood. To address this, we examined and compared changes in wood undergoing decay by two species of brown rot fungi, Gloeophyllum trabeum and Meruliporia incrassata, and two species of white rot fungi, Irpex lacteus and Pycnoporus sanguineus, using X-ray diffraction (XRD) and 13C solid-state nuclear magnetic resonance (NMR) spectroscopy. The overall percent crystallinity in wood undergoing decay by M. incrassata, G. trabeum, and I. lacteus appeared to decrease according to the stage of decay, while in wood decayed by P. sanguineus the crystallinity was found to increase during some stages of degradation. This result is suggested to be potentially due to the different decay strategies employed by these fungi. The average spacing between the 200 cellulose crystal planes was significantly decreased in wood degraded by brown rot, whereas changes observed in wood degraded by the two white rot fungi examined varied according to the selectivity for lignin. The conclusions were supported by a quantitative analysis of the structural components in the wood before and during decay confirming the distinct differences observed for brown and white rot fungi. The results from this study were consistent with differences in degradation methods previously reported among fungal species, specifically more non-enzymatic degradation in brown rot versus more enzymatic degradation in white rot.
► The brown rot fungi decreased the percent crystallinity. ► Simultaneous white rot fungus similarly decreased the percent crystallinity. ► Selective white rot fungus was found to increase the percent crystallinity. ► Changes in d-spacing were species dependent. ► Variation was observed in lignin structures after decay.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The effect of hot-water extraction and two types of fungal decay, brown rot and white rot, on wood crystalline cellulose structure was examined using a combination of X-ray diffraction (XRD) and
13
C ...solid-state nuclear magnetic resonance (NMR) spectroscopy. Although having opposite effects on the overall crystallinity of the wood, the XRD results revealed that both extraction and brown-rot decay caused a significant decrease in the 200 crystal plane spacing (
d
-spacing) not seen for the white-rotted samples. This effect was found to be additive, as samples that were first extracted, then decayed showed a double decrease in
d
-spacing compared to that caused by extraction alone. This suggested that, despite having a similarly directed effect on the spacing of the crystalline planes, the two treatment methods facilitate a decrease in
d
-spacing in different ways. NMR results support the conclusion of differing structural effects, suggesting that the hot-water extraction procedure was causing a co-crystallization of existing crystalline domains, while the brown rot decay was depolymerizing the cellulose chains of the crystals, possibly allowing the remaining crystalline material the freedom to relax into a more energetically favorable, tightly packed state. These findings could have important implications for those seeking to understand the effects of modification treatments or biodegradation of crystalline cellulose nanostructures in their native states.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The depletion of root-available Ca in northern forest soils exposed to decades of increased acid deposition adversely affects forest health and productivity. Laboratory studies indicated the ...potential of wood-decay fungi to restore lost Ca. This study presents changes in concentration of Ca, Mg, and K in sapwood of red spruce (Picea rubens Sarg.), red maple (Acer rubrum L.), eastern hemlock (Tsuga canadensis (L.) Carrière), and paper birch (Betula papyrifera Marshall) during the decay process at two experimental forests for 12 years and to compare concentrations of exchangeable Ca, Mg, and Al in decayed wood residues at 10 and 12 years with those in the forest floor. Significant loss of mass indicated by decreasing wood density occurred after 2â8 years in conifers and after only 2 years in hardwoods. A significant gain in wood K was observed at 2 years followed by a significant loss at 8 years. A negligible gain in Ca concentration occurred at 2 years and a substantial gain at 8 years. Observed changes in Mg concentration were variable. No significant difference in exchangeable Ca concentration was observed between decayed wood residue of spruce and maple and the forest floor. However, decayed wood residue had a much lower Al concentration and molar Al/Ca ratio, a condition characteristic of sites with high root-available Ca.
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BF, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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