Complex waste stream valorization through combined enzymatic hydrolysis and catabolic assimilation by Pseudomonas putida.

Biogenic waste-derived feedstocks for production of fuels, chemicals, and materials offer great potential supporting the transition to net-zero and greater circularity. However, such feedstocks are heterogeneous and subject to geographical and seasonal variability. Here, we show that, through careful strain selection and metabolic engineering, Pseudomonas putida can be employed to permit efficient co-utilization of highly heterogeneous substrate compositions derived from hydrolyzed mixed municipal-like waste fractions (food, plastic, organic, paper, cardboard, and textiles) for growth and synthesis of exemplar bioproducts.

Live cell imaging of β-tubulin mRNA reveals spatiotemporal expression dynamics in the filamentous fungus Aspergillus oryzae.

In filamentous fungi, microtubules are important for polar growth and morphological maintenance and serve as rails for intracellular trafficking. The molecular mechanisms associated with microtubules have been analyzed. However, little is known about when and where tubulin, a component of microtubules, is biosynthesized in multinuclear and multicellular filamentous fungi.

Development of High-Performance Whole Cell Biosensors Aided by Statistical Modeling.

Whole cell biosensors are genetic systems that link the presence of a chemical, or other stimulus, to a user-defined gene expression output for applications in sensing and control. However, the gene expression level of biosensor regulatory components required for optimal performance is nonintuitive, and classical iterative approaches do not efficiently explore multidimensional experimental space. To overcome these challenges, we used a design of experiments (DoE) methodology to efficiently map gene expression levels and provide biosensors with enhanced performance.

Rational engineering of photosynthetic electron flux enhances light-powered cytochrome P450 activity.

In this study, we exploited a modified photosynthetic electron transfer chain (PET) in the model cyanobacterium PCC 7002, where electrons derived from water-splitting are used to power reactions catalyzed by a heterologous cytochrome P450 (CYP1A1). A simple fluorescent assay for CYP1A1 activity was employed to determine the impact of rationally engineering of photosynthetic electron flow. This showed that knocking out a subunit of the type I NADH dehydrogenase complex (NDH-1), suggested to be involved in cyclic photosynthetic electron flow (Δ), can double the activity of CYP1A1, with a concomitant increase in the flux of electrons from photosynthesis.

A new proteinaceous pathogen-associated molecular pattern (PAMP) identified in Ascomycete fungi induces cell death in Solanaceae.

Pathogen-associated molecular patterns (PAMPs) are detected by plant pattern recognition receptors (PRRs), which gives rise to PAMP-triggered immunity (PTI). We characterized a novel fungal PAMP, Cell Death Inducing 1 (RcCDI1), identified in the Rhynchosporium commune transcriptome sampled at an early stage of barley (Hordeum vulgare) infection. The ability of RcCDI1 and its homologues from different fungal species to induce cell death in Nicotiana benthamiana was tested following agroinfiltration or infiltration of recombinant proteins produced by Pichia pastoris.

Tapping the Unused Potential of Photosynthesis with a Heterologous Electron Sink.

Increasing the efficiency of the conversion of light energy to products by photosynthesis represents a grand challenge in biotechnology. Photosynthesis is limited by the carbon-fixing enzyme Rubisco resulting in much of the absorbed energy being wasted as heat or fluorescence or lost as excess reductant via alternative electron dissipation pathways. To harness this wasted reductant, we engineered the model cyanobacterium Synechococcus PCC 7002 to express the mammalian cytochrome P450 CYP1A1 to serve as an artificial electron sink for excess electrons derived from light-catalyzed water-splitting.

Horizontal Gene Transfer from Bacteria Has Enabled the Plant-Parasitic Nematode Globodera pallida to Feed on Host-Derived Sucrose.

The evolution of plant-parasitic nematodes (PPN) is unusual in that these organisms have acquired a range of genes from bacteria via horizontal gene transfer (HGT). The proteins encoded by most of these genes are involved in metabolism of various components of the plant cell wall during invasion of the host. Recent genome sequencing projects for PPN have shown that Glycosyl Hydrolase Family 32 (GH32) sequences are present in several PPN species.

Hook is an adapter that coordinates kinesin-3 and dynein cargo attachment on early endosomes.

Bidirectional membrane trafficking along microtubules is mediated by kinesin-1, kinesin-3, and dynein. Several organelle-bound adapters for kinesin-1 and dynein have been reported that orchestrate their opposing activity. However, the coordination of kinesin-3/dynein-mediated transport is not understood.

Septins are important for cell polarity, septation and asexual spore formation in Neurospora crassa and show different patterns of localisation at germ tube tips.

Septins are GTP-binding cytoskeletal proteins that contribute to cell polarity, vesicle trafficking, cytokinesis and cell morphogenesis. Here we have characterised the six septins encoded by the genome of the model filamentous fungus Neurospora crassa. Analysis of septin null mutants demonstrated that septins limit the sites of emergence of germ tubes and are important for septation and conidiation in N.

Actin organization and dynamics in filamentous fungi.

Growth and morphogenesis of filamentous fungi is underpinned by dynamic reorganization and polarization of the actin cytoskeleton. Actin has crucial roles in exocytosis, endocytosis, organelle movement and cytokinesis in fungi, and these processes are coupled to the production of distinct higher-order structures (actin patches, cables and rings) that generate forces or serve as tracks for intracellular transport. New approaches for imaging actin in living cells are revealing important similarities and differences in actin architecture and organization within the fungal kingdom, and have yielded key insights into cell polarity, tip growth and long-distance intracellular transport.

Form follows function -- the versatile fungal cytoskeleton.

The cytoskeleton plays a major role in the regulation of fungal cell morphogenesis. The fungal cytoskeleton is comprised of three polymers: F-actin, microtubules and septins. Due to the successful application of the newly developed Lifeact probe for live-cell imaging of F-actin it is now possible, in combination with existing microtubule markers and fluorescently labelled septins, to monitor real-time dynamics of the entire fungal cytoskeleton, and reassess the many and integrated roles of F-actin, microtubules and septins throughout fungal growth and development.

A novel family of dehydrin-like proteins is involved in stress response in the human fungal pathogen Aspergillus fumigatus.

During a search for genes controlling conidial dormancy in Aspergillus fumigatus, two dehydrin-like genes, DprA and DprB, were identified. The deduced proteins had repeated stretches of 23 amino acids that contained a conserved dehydrin-like protein (DPR) motif. Disrupted DprAΔ mutants were hypersensitive to oxidative stress and to phagocytic killing, whereas DprBΔ mutants were impaired in osmotic and pH stress responses.

F-actin dynamics in Neurospora crassa.

This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N.