Design and Construction of an Experimental Setup to Enhance Mineral Weathering through the Activity of Soil Organisms.

Enhanced weathering (EW) is an emerging carbon dioxide (CO2) removal technology that can contribute to climate change mitigation. This technology relies on accelerating the natural process of mineral weathering in soils by manipulating the abiotic variables that govern this process, in particular mineral grain size and exposure to acids dissolved in water. EW mainly aims at reducing atmospheric CO2 concentrations by enhancing inorganic carbon sequestration.

A Conserved Second Sphere Residue Tunes Copper Site Reactivity in Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are powerful monocopper enzymes that can activate strong C-H bonds through a mechanism that remains largely unknown. Herein, we investigated the role of a conserved glutamine/glutamate in the second coordination sphere. Mutation of the Gln in AA9C to Glu, Asp, or Asn showed that the nature and distance of the headgroup to the copper fine-tune LPMO functionality and copper reactivity.

Bidirectional Promoter Libraries Enable the Balanced Co-expression of Two Target Genes in E. coli.

In this chapter, we present a bidirectional promoter library toolbox to evaluate fast and efficiently the optimal conditions for the balanced co-expression of two target genes. As a proof-of-concept, we demonstrate the co-expression of CYP505x and the GroEL/ES complex, which resulted in noticeably elevated enzyme activity with one of the de-novo-designed promoters of the library. The new toolbox offers a straightforward one-pot cloning approach and is highly modular.

Fast and Specific Peroxygenase Reactions Catalyzed by Fungal Mono-Copper Enzymes.

The copper-dependent lytic polysaccharide monooxygenases (LPMOs) are receiving attention because of their role in the degradation of recalcitrant biomass and their intriguing catalytic properties. The fundamentals of LPMO catalysis remain somewhat enigmatic as the LPMO reaction is affected by a multitude of LPMO- and co-substrate-mediated (side) reactions that result in a complex reaction network. We have performed kinetic studies with two LPMOs that are active on soluble substrates, AA9C and AA9A, using various reductants typically employed for LPMO activation.

Kinetic Characterization of a Putatively Chitin-Active LPMO Reveals a Preference for Soluble Substrates and Absence of Monooxygenase Activity.

Enzymes known as lytic polysaccharide monooxygenases (LPMOs) are recognized as important contributors to aerobic enzymatic degradation of recalcitrant polysaccharides such as chitin and cellulose. LPMOs are remarkably abundant in nature, with some fungal species possessing more than 50 LPMO genes, and the biological implications of this diversity remain enigmatic. For example, chitin-active LPMOs have been encountered in biological niches where chitin conversion does not seem to take place.

Novel molecular biological tools for the efficient expression of fungal lytic polysaccharide monooxygenases in Pichia pastoris.

Background: Lytic polysaccharide monooxygenases (LPMOs) are attracting large attention due their ability to degrade recalcitrant polysaccharides in biomass conversion and to perform powerful redox chemistry.

Results: We have established a universal Pichia pastoris platform for the expression of fungal LPMOs using state-of-the-art recombination cloning and modern molecular biological tools to achieve high yields from shake-flask cultivation and simple tag-less single-step purification. Yields are very favorable with up to 42 mg per liter medium for four different LPMOs spanning three different families.