Finnigan, W. | bioRxiv Science

bioRxiv2020

The effect of terminal globular domains on the response of recombinant mini-spidroins to fiber spinning triggers

Spider silk spidroins consist of long repetitive protein strands, flanked by globular terminal domains. The globular domains are often omitted in recombinant spidroins, but are thought to be essential for the spiders natural spinning process. Mimicking this spinning process could be an essential step towards producing strong synthetic spider silk. Here we describe the production of a range of mini-spidroins with both terminal domains, and characterize their response to a number of biomimetic spinning triggers. Our results suggest that the inclusion of the terminal domains is needed to match the response to shear that native spidroins exhibit. Our results also suggest that a pH drop alone is insufficient to trigger assembly in a wet-spinning process, and must be combined with salting-out for effective fiber formation. With these insights, we applied these assembly triggers for relatively biomimetic wet spinning. This work adds to the foundation of literature for developing improved biomimetic spinning techniques, which ought to result in synthetic silk that more closely approximates the unique properties of native spider silk.

bioengineering↗

bioRxiv2019

Engineering a seven enzyme biotransformation using mathematical modelling and characterized enzyme parts

Multi-step enzyme reactions offer considerable cost and productivity benefits. Process models offer a route to understanding the complexity of these reactions, and allow for their optimization. Despite the increasing prevalence of multi-step biotransformations, there are few examples of process models for enzyme reactions. From a toolbox of characterized enzyme parts, we demonstrate the construction of a process model for a seven enzyme, three step biotransformation using isolated enzymes. Enzymes for cofactor regeneration were employed to make this in vitro reaction economical. Good modelling practice was critical in evaluating the impact of approximations and experimental error. We show that the use and validation of process models was instrumental in realizing and removing process bottlenecks, identifying divergent behavior, and for the optimization of the entire reaction using a genetic algorithm. We validated the optimized reaction to demonstrate that complex multi-step reactions with cofactor recycling involving at least seven enzymes can be reliably modelled and optimized.\n\nSignificance statementThis study examines the challenge of modeling and optimizing multi-enzyme cascades. We detail the development, testing and optimization of a deterministic model of a three enzyme cascade with four cofactor regeneration enzymes. Significantly, the model could be easily used to predict the optimal concentrations of each enzyme in order to get maximum flux through the cascade. This prediction was strongly validated experimentally. The success of our model demonstrates that robust models of systems of at least seven enzymes are readily achievable. We highlight the importance of following good modeling practice to evaluate model quality and limitations. Examining deviations from expected behavior provided additional insight into the model and enzymes. This work provides a template for developing larger deterministic models of enzyme cascades.

biochemistry↗