Recombinant sea urchin vascular endothelial growth factor directs single-crystal growth and branching in vitro.

Biomineralization in sea urchin embryos is a crystal growth process that results in oriented single-crystalline spicules with a complex branching shape and smoothly curving surfaces. Uniquely, the primary mesenchyme cells (PMCs) that construct the endoskeleton can be cultured in vitro. However, in the absence of morphogenetic cues secreted by other cells in the embryo, spicules deposited in PMC culture lack the complex branching behavior observed in the embryo. Herein we demonstrate that recombinant sea urchin vascular endothelial growth factor (rVEGF), a signaling molecule that interacts with a cell-surface receptor, induces spiculogenesis and controls the spicule shape in PMC culture. Depending on the rVEGF concentration, PMCs deposit linear, "h"- and "H"-shaped, or triradiate spicules. Remarkably, the change from linear to triradiate occurs with a switch from bidirectional crystal growth parallel to the calcite c axis to growth along the three a axes. This finding has implications for our understanding of how cells integrate morphogenesis on the multi-micrometer scale with control over lattice orientation on the atomic scale. The PMC model system is uniquely suited to investigate this mechanism and develop biotechnological approaches to single-crystal growth.

High-throughput optofluidic screening for improved microbial cell factories via real-time micron-scale productivity monitoring.

The industrial synthetic biology sector has made huge investments to achieve relevant miniaturized screening systems for scalable fermentation. Here we present the first example of a high-throughput (>103 genotypes per week) perfusion-based screening system to improve small-molecule secretion from microbial strains. Using the Berkeley Lights Beacon® system, the productivity of each strain could be directly monitored in real time during continuous culture, yielding phenotypes that correlated strongly (r2 > 0.8, p < 0.0005) with behavior in industrially relevant bioreactor processes. This method allows a much closer approximation of a typical fed-batch fermentation than conventional batch-like droplet or microplate culture models, in addition to rich time-dependent data on growth and productivity. We demonstrate these advantages by application to the improvement of high-productivity strains using whole-genome random mutagenesis, yielding mutants with substantially improved (by up to 85%) peak specific productivities in bioreactors. Each screen of ∼5 × 103 mutants could be completed in under 8 days (including 5 days involving user intervention), saving ∼50-75% of the time required for conventional microplate-based screening methods.

Characterizing Isozymes of Chlorite Dismutase for Water Treatment.

This work investigated the potential for biocatalytic degradation of micropollutants, focusing on chlorine oxyanions as model contaminants, by mining biology to identify promising biocatalysts. Existing isozymes of chlorite dismutase (Cld) were characterized with respect to parameters relevant to this high volume, low-value product application: kinetic parameters, resistance to catalytic inactivation, and stability. Maximum reaction velocities (Vmax) were typically on the order of 104 µmol min-1 (µmol heme)-1. Substrate affinity (Km) values were on the order of 100 µM, except for the Cld from Candidatus Nitrospira defluvii (NdCld), which showed a significantly lower affinity for chlorite. NdCld also had the highest susceptibility to catalytic inactivation. In contrast, the Cld from Ideonella dechloratans was least susceptible to catalytic inactivation, with a maximum turnover number of approximately 150,000, more than sevenfold higher than other tested isozymes. Under non-reactive conditions, Cld was quite stable, retaining over 50% of activity after 30 days, and most samples retained activity even after 90-100 days. Overall, Cld from I. dechloratans was the most promising candidate for environmental applications, having high affinity and activity, a relatively low propensity for catalytic inactivation, and excellent stability.