DisCoTune: versatile auxiliary plasmids for the production of disulphide-containing proteins and peptides in the E. coli T7 system.

Secreted proteins and peptides hold large potential both as therapeutics and as enzyme catalysts in biotechnology. The high stability of many secreted proteins helps maintain functional integrity in changing chemical environments and is a contributing factor to their commercial potential. Disulphide bonds constitute an important post-translational modification that stabilizes many of these proteins and thus preserves the active state under chemically stressful conditions. Despite their importance, the discovery and applications within this group of proteins and peptides are limited by the availability of synthetic biology tools and heterologous production systems that allow for efficient formation of disulphide bonds. Here, we refine the design of two DisCoTune (Disulphide bond formation in E. coli with tunable expression) plasmids that enable the formation of disulphides in the highly popular Escherichia coli T7 protein production system. We show that this new system promotes significantly higher yield and activity of an industrial protease and a conotoxin, which belongs to a group of disulphide-rich venom peptides from cone snails with strong potential as research tools and pharmacological agents.

LyGo: A Platform for Rapid Screening of Lytic Polysaccharide Monooxygenase Production.

Environmentally friendly sources of energy and chemicals are essential constituents of a sustainable society. An important step toward this goal is the utilization of biomass to supply building blocks for future biorefineries. Lytic polysaccharide monooxygenases (LPMOs) are enzymes that play a critical role in breaking the chemical bonds in the most abundant polymers found in recalcitrant biomass, such as cellulose and chitin. To use them in industrial processes they need to be produced in high titers in cell factories. Predicting optimal strategies for producing LPMOs is often nontrivial, and methods allowing for screening several strategies simultaneously are therefore needed. Here, we present a standardized platform for cloning LPMOs. The platform allows users to combine gene fragments with 14 different expression vectors in a simple 15 min reaction, thus enabling rapid exploration of several gene contexts, hosts, and expression strategies in parallel. The open-source LyGo platform is accompanied by easy-to-follow online protocols for both cloning and expression. As a demonstration of its utility, we explore different strategies for expressing several different LPMOs in Escherichia coli, Bacillus subtilis, and Komagataella phaffii.

Copper binding and reactivity at the histidine brace motif: insights from mutational analysis of the Pseudomonas fluorescens copper chaperone CopC.

The histidine brace (His-brace) is a copper-binding motif that is associated with both oxidative enzymes and proteinaceous copper chaperones. Here, we used biochemical and structural methods to characterize mutants of a His-brace-containing copper chaperone from Pseudomonas fluorescens (PfCopC). A total of 15 amino acid variants in primary and second-sphere residues were produced and characterized in terms of their copper binding and redox properties. PfCopC has a very high affinity for Cu(II) and also binds Cu(I). A high reorganization barrier likely prevents redox cycling and, thus, catalysis. In contrast, mutations in the conserved second-sphere Glu27 enable slow oxidation of ascorbate. The crystal structure of the variant E27A confirmed copper binding at the His-brace. Unexpectedly, Asp83 at the equatorial position was shown to be indispensable for Cu(II) binding in the His-brace of PfCopC. A PfCopC mutant that was designed to mimic the His-brace from lytic polysaccharide monooxygenase-like family X325 did not bind Cu(II), but was still able to bind Cu(I). These results highlight the importance of the proteinaceous environment around the copper His-brace for reactivity and, thus, the difference between enzyme and chaperone.

Colorimetric LPMO assay with direct implication for cellulolytic activity.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) are important industrial enzymes known for their catalytic degradation of recalcitrant polymers such as cellulose or chitin. Their activity can be measured by lengthy HPLC methods, while high-throughput methods are less specific. A fast and specific LPMO assay would simplify screening for new or engineered LPMOs and accelerate biochemical characterization. RESULTS: A novel LPMO activity assay was developed based on the production of the dye phenolphthalein (PHP) from its reduced counterpart (rPHP). The colour response of rPHP oxidisation catalysed by the cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A), was found to increase tenfold by adding dehydroascorbate (DHA) as a co-substrate. The assay using a combination of rPHP and DHA was tested on 12 different metallo-enzymes, but only the LPMOs catalysed this reaction. The assay was optimized for characterization of TaAA9A and showed a sensitivity of 15 nM after 30 min incubation. It followed apparent Michaelis-Menten kinetics with kcat = 0.09 s-1 and KM = 244 µM, and the assay was used to confirm stoichiometric copper-enzyme binding and enzyme unfolding at a temperature of approximately 60 °C. DHA, glutathione and fructose were found to enhance LPMO oxidation of rPHP and in the optimized assay conditions these co-substrates also enabled cellulose degradation. CONCLUSIONS: This novel and specific LPMO assay can be carried out in a convenient microtiter plate format ready for high-throughput screening and enzyme characterization. DHA was the best co-substrate tested for oxidation of rPHP and this preference appears to be LPMO-specific. The identified co-substrates DHA and fructose are not normally considered as LPMO co-substrates but here they are shown to facilitate both oxidation of rPHP and degradation of cellulose. This is a rare example of a finding from a high-throughput assay that directly translate into enzyme activity on an insoluble substrate. The rPHP-based assay thus expands our understanding of LPMO catalysed reactions and has the potential to characterize LPMO activity in industrial settings, where usual co-substrates such as ascorbate and oxygen are depleted.

The three-dimensional structure of an H-superfamily conotoxin reveals a granulin fold arising from a common ICK cysteine framework.

Venomous marine cone snails produce peptide toxins (conotoxins) that bind ion channels and receptors with high specificity and therefore are important pharmacological tools. Conotoxins contain conserved cysteine residues that form disulfide bonds that stabilize their structures. To gain structural insight into the large, yet poorly characterized conotoxin H-superfamily, we used NMR and CD spectroscopy along with MS-based analyses to investigate H-Vc7.2 from Conus victoriae, a peptide with a VI/VII cysteine framework. This framework has CysI-CysIV/CysII-CysV/CysIII-CysVI connectivities, which have invariably been associated with the inhibitor cystine knot (ICK) fold. However, the solution structure of recombinantly expressed and purified H-Vc7.2 revealed that although it displays the expected cysteine connectivities, H-Vc7.2 adopts a different fold consisting of two stacked ß-hairpins with opposing ß-strands connected by two parallel disulfide bonds, a structure homologous to the N-terminal region of the human granulin protein. Using structural comparisons, we subsequently identified several toxins and nontoxin proteins with this "mini-granulin" fold. These findings raise fundamental questions concerning sequence-structure relationships within peptides and proteins and the key determinants that specify a given fold.