MreB Filaments in the Elongasome Modulate E. coli Membrane Curvature.

MreB, a bacterial actin homolog, plays a pivotal role in defining the shape of rod-shaped bacteria by coordinating peptidoglycan synthesis during cell elongation. It forms filaments that interact with the cytosolic leaflet of the cell membrane, as well as with membrane proteins and other cytosolic proteins. In this study, molecular dynamics (MD) simulations were used to investigate the interactions between Escherichia coli (E.

Scaling-up the Bioconversion of Lignin to 2,4-Pyridinedicarboxylic Acid With Engineered Pseudomonas putida for Bio-Based Plastics Production.

2,4-pyridinedicarboxylic acid (PDCA) is a promising bio-based compound to substitute petroleum-derived terephthalic acid in plastics. It is produced through the microbial conversion of lignin substrates with engineered microorganisms like Pseudomonas putida. To this point, an efficient bioproduction process for PDCA has not yet been established.

Degradation of brown coal and 1-methylnaphthalene by a Micrococcus luteus isolate: investigation of a novel aromatic degradation gene cluster containing paa genes.

Aims: To identify novel brown coal-degrading bacteria, and elucidate the biochemical pathways involved in brown coal degradation.

Methods And Results: Four bacterial isolates were identified from the surface of Indonesian brown coal, which can utilize naphthalene and 1-methylnaphthalene as carbon sources for growth. The genome sequence of the best-performing Micrococcus luteus K1 strain was determined.

Assessing iron reductase activity: Methodologies for fungal degradation of lignocellulose.

Lignocellulosic biomass (LCB) represents a critical renewable resource for bioenergy and bioproducts, yet its efficient degradation is constrained by the recalcitrance of lignin. This study evaluates the methodologies for assessing iron reductase activity of recombinant proteins IR1 and IR2 from brown rot fungi Serpula lacrymans, emphasizing their capacity to degrade lignocellulose. Iron plays a crucial role in wood decay, by functioning within the active sites of ligninolytic enzymes and fostering the production of reactive oxygen species (ROS) through iron-binding compounds.

Glycolate oxidase from Rhodococcus jostii RHA1: An accessory enzyme for lignin bioconversion with bacterial DyP-type peroxidases.

Accessory enzymes have been identified in lignin-degrading fungi and bacteria that can generate hydrogen peroxide, which is used as a co-substrate by lignin-oxidising peroxidases. This article describes a glycolate oxidase enzyme from lignin-degrading bacterium Rhodococcus jostii RHA1, which functions as an efficient accessory enzyme for degradation of polymeric lignin substrates by bacterial DyP-type peroxidases. The article describes: (1) enzyme purification; (2) assays for enzyme activity; (3) analysis of substrate specificity; (4) assays for enzyme combinations with bacterial DyP-type peroxidases; (5) analysis of low molecular weight products obtained using enzyme combinations.

Dye-decolorising peroxidase DyP1B from Pseudomonas fluorescens: Expression, reconstitution and reaction with polymeric lignin substrates.

Several bacterial dye-decolorising peroxidases have been identified, that have activity for oxidation of lignin model compounds and polymeric lignin. This article describes biochemical methods for lignin-oxidising peroxidase DyP1B from Pseudomonas fluorescens Pf-5. The article presents methods for: (1) enzyme purification and heme reconstitution; (2) steady-state kinetic enzyme assays; (3) pre-steady state kinetic analysis; (4) testing of the enzyme against polymeric lignin substrates.

Assay methods and colorimetric screens for lignin-degrading microbes and lignin-oxidising enzymes.

Assaying enzymes and microbes for activity for degradation of polymeric lignin is inherently challenging to do. This article describes several methods that our research group has developed for assay of lignin-oxidising enzymes and lignin-degrading microbes. The assay methods involve (1) colorimetric assays involving chemically nitrated lignin; (2) changes in molecular weight using gel filtration chromatography; (3) delignification of lignocellulose using Klason assay; (4) colorimetric assays for release of low molecular weight phenols and carbonyl compounds.

Antimicrobial triazinedione inhibitors of the translocase MraY-protein E interaction site: synergistic effects with bacitracin imply a new mechanism of action.

translocase MraY is the target for bacteriolytic protein E from bacteriophage ϕX174, interacting at a site close to Phe-288 on helix 9, on the extracellular face of the protein. A peptide motif Arg-Trp-x-x-Trp from protein E was used to design a set of triazinedione peptidomimetics, which inhibit particulate MraY (6d IC 48 μM), and show antimicrobial activity against Gram-negative and Gram-positive antibiotic-resistant clinical strains (7j MIC 16 μg mL, MRSA 2-4 μg mL). Docking against a predicted structure for MraY revealed two possible binding sites close to helix 9, the binding site for protein E.

Sustainable production of aromatic chemicals from lignin using enzymes and engineered microbes.

Lignin is an aromatic biopolymer found in plant cell walls and is the most abundant source of renewable aromatic carbon in the biosphere. Hence there is considerable interest in the conversion of lignin, either derived from agricultural waste or produced as a byproduct of pulp/paper manufacture, into high-value chemicals. Although lignin is rather inert, due to the presence of ether C-O and C-C linkages, several microbes are able to degrade lignin.

Pharmaceutical applications of lignin-derived chemicals and lignin-based materials: linking lignin source and processing with clinical indication.

Lignocellulosic biomass is one of the most abundant bioresources on Earth. Over recent decades, various valorisation techniques have been developed to produce value-added products from the cellulosic and hemicellulosic fractions of this biomass. Lignin is the third major component accounting for 10-30% (w/w).

In vitro assay and inhibition of 9-cis-epoxycarotenoid dioxygenase (NCED) from Solanum lycopersicum and Zea mays.

The article reports methods for the expression and assay of 9-cis-epoxycarotenoid cleavage dioxygenase (NCED), an enzyme involved in the biosynthesis of phytohormone abscisic acid in plants. A method for the preparation of the unstable substrate 9'-cis-neoxanthin from fresh spinach is described. The inhibition of Solanum lycopersicum NCED by a series of aryl hydroxamic acid inhibitors is illustrated, and inhibitors D2 and D4 are assayed against NCED isozymes from Zea mays.

Ether Bond Cleavage of a Phenylcoumaran β-5 Lignin Model Compound and Polymeric Lignin Catalysed by a LigE-type Etherase from Agrobacterium sp.

A LigE-type beta-etherase enzyme from lignin-degrading Agrobacterium sp. has been identified, which assists degradation of polymeric lignins. Testing against lignin dimer model compounds revealed that it does not catalyse the previously reported reaction of Sphingobium SYK-6 LigE, but instead shows activity for a β-5 phenylcoumaran lignin dimer.

The chemical logic of enzymatic lignin degradation.

Lignin is an aromatic heteropolymer, found in plant cell walls as 20-30% of lignocellulose. It represents the most abundant source of renewable aromatic carbon in the biosphere, hence, if it could be depolymerised efficiently, then it would be a highly valuable source of renewable aromatic chemicals. However, lignin presents a number of difficulties for biocatalytic or chemocatalytic breakdown.

Overexpression of endogenous multi-copper oxidases mcoA and mcoC in Rhodococcus jostii RHA1 enhances lignin bioconversion to 2,4-pyridine-dicarboxylic acid.

To improve the titre of lignin-derived pyridine-dicarboxylic acid (PDCA) products in engineered Rhodococcus jostii RHA1 strains, plasmid-based overexpression of seven endogenous and exogenous lignin-degrading genes was tested. Overexpression of endogenous multi-copper oxidases mcoA, mcoB, and mcoC was found to enhance 2,4-PDCA production by 2.5-, 1.

Engineered co-culture for consolidated production of phenylpropanoids directly from aromatic-rich biomass.

Consolidated bioprocesses for the in situ hydrolysis and conversion of biomass feedstocks into value-added products offers great potential for both process and cost reduction. However, to date few consolidated bioprocesses have been developed that target aromatic rich feedstock fractions. Reported here is the development of synthetic co-cultivation for the consolidated hydrolysis and valorisation of corncob hydroxycinnamic acids.

Engineering of RHA1 for utilisation of carboxymethylcellulose.

RHA1 was engineered to utilise the cellulose component of lignocellulose, as well as the lignin fraction, by introduction of cellulase genes. The genome of RHA1 was found to contain two β-glucosidase genes, RHA1_ro01034 and RHA1_ro02947, which support growth on cellobiose as growth substrate. Five Gram-positive endocellulase genes and one exocellulase gene were cloned into expression vector pTipQC2, and expressed in RHA1.

Sustainable biosynthetic pathways to value-added bioproducts from hydroxycinnamic acids.

The biorefinery concept, in which biomass is utilized for the production of fuels and chemicals, emerges as an eco-friendly, cost-effective, and renewable alternative to petrochemical-based production. The hydroxycinnamic acid fraction of lignocellulosic biomass represents an untapped source of aromatic molecules that can be converted to numerous high-value products with industrial applications, including in the flavor and fragrance sector and pharmaceuticals. This review describes several biochemical pathways useful in the development of a biorefinery concept based on the biocatalytic conversion of the hydroxycinnamic acids ferulic, caffeic, and p-coumaric acid into high-value molecules.

Metatranscriptomic analysis of the gut microbiome of black soldier fly larvae reared on lignocellulose-rich fiber diets unveils key lignocellulolytic enzymes.

Recently, interest in the black soldier fly larvae (BSFL) gut microbiome has received increased attention primarily due to their role in waste bioconversion. However, there is a lack of information on the positive effect on the activities of the gut microbiomes and enzymes (CAZyme families) acting on lignocellulose. In this study, BSFL were subjected to lignocellulose-rich diets: chicken feed (), chicken manure (CM), brewers' spent grain (BSG), and water hyacinth (WH).

Application of RHA1 glycolate oxidase as an efficient accessory enzyme for lignin conversion by bacterial Dyp peroxidase enzymes.

Lignin oxidation by bacterial dye-decolorizing peroxidase enzymes requires hydrogen peroxide as a co-substrate, an unstable and corrosive oxidant. We have identified a glycolate oxidase enzyme from RHA1 that can couple effectively at pH 6.5 with DyP peroxidase enzymes from sp.

Elucidation of microbial lignin degradation pathways using synthetic isotope-labelled lignin.

Pathways by which the biopolymer lignin is broken down by soil microbes could be used to engineer new biocatalytic routes from lignin to renewable chemicals, but are currently not fully understood. In order to probe these pathways, we have prepared synthetic lignins containing C at the sidechain β-carbon. Feeding of [β-C]-labelled DHP lignin to RHA1 has led to the incorporation of C label into metabolites oxalic acid, 4-hydroxyphenylacetic acid, and 4-hydroxy-3-methoxyphenylacetic acid, confirming that they are derived from lignin breakdown.

Characterisation of an unusual cysteine pair in the Rieske carnitine monooxygenase CntA catalytic site.

Rieske monooxygenases undertake complex catalysis integral to marine, terrestrial and human gut-ecosystems. Group-I to -IV Rieske monooxygenases accept aromatic substrates and have well-characterised catalytic mechanisms. Nascent to our understanding are Group-V members catalysing the oxidation/breakdown of quaternary ammonium substrates.

Periplasmic expression of Pseudomonas fluorescens peroxidase Dyp1B and site-directed mutant Dyp1B enzymes enhances polymeric lignin degradation activity in Pseudomonas putida KT2440.

Expression of lignin-oxidising Pseudomonas fluorescens Dyp1B in the periplasm of Pseudomonas putida KT2440, using a tat fusion construct, was found to lead to enhanced whole cell activity for oxidation of DCP and polymeric lignin substrates. Four amino acid residues predicted to lie at the manganese ion binding site of Pseudomonas fluorescens peroxidase Dyp1B were investigated using site-directed mutagenesis. Mutants H127R and S223A showed 2-fold and 4-fold higher k for Mn(II) oxidation respectively, and mutant S223A showed 2-fold enhanced production of low molecular weight phenolic products from a polymeric soda lignin.

Bioconversion of lignin-derived aromatics into the building block pyridine 2,4-dicarboxylic acid by engineering recombinant Pseudomonas putida strains.

2,4 pyridine dicarboxylic acid (2,4 PDCA) is an analogue of terephthalate, and hence a target chemical in the field of bio-based plastics. Here, Pseudomonas putida KT2440 strains were engineered to efficiently drive the metabolism of lignin-derived monoaromatics towards 2,4 PDCA in a resting cells-based bioprocess that alleviates growth-coupled limitations and allows biocatalysts recycling. Native β-ketoadipate pathway was blocked by replacing protocatechuate 3,4-dioxygenase by the exogenous LigAB extradiol dioxygenase.

Peptidomimetic analogues of an Arg-Trp-x-x-Trp motif responsible for interaction of translocase MraY with bacteriophage ϕX174 lysis protein E.

Translocase MraY is the target for bacteriophage ϕX174 lysis protein E, which interacts via a protein-protein interaction mediated by Phe-288 and Glu-287 of E. coli MraY, and an Arg-Trp-x-x-Trp motif on protein E, also found in several cationic antimicrobial peptides. Analogues of Arg-Trp-octyl ester, found previously to show antimicrobial activity, were tested for antimicrobial activity, with Lys-Trp-oct (MICP.

Metabolic engineering of Rhodococcus jostii RHA1 for production of pyridine-dicarboxylic acids from lignin.

Genetic modification of Rhodococcus jostii RHA1 was carried out in order to optimise the production of pyridine-2,4-dicarboxylic acid and pyridine-2,5-dicarboxylic acid bioproducts from lignin or lignocellulose breakdown, via insertion of either the Sphingobium SYK-6 ligAB genes or Paenibacillus praA gene respectively. Insertion of inducible plasmid pTipQC2 expression vector containing either ligAB or praA genes into a ΔpcaHG R. jostii RHA1 gene deletion strain gave 2-threefold higher titres of PDCA production from lignocellulose (200-287 mg/L), compared to plasmid expression in wild-type R.