A high-throughput expression and screening platform for applications-driven PETase engineering.

The environmental consequences of plastic waste have impacted all kingdoms of life in terrestrial and aquatic ecosystems. However, as the burden of plastic pollution has increased, microbes have evolved to utilize anthropogenic polymers as nutrient sources. Of depolymerase enzymes, the best characterized is PETase, which hydrolyzes aromatic polyesters. PETase engineering has made impressive progress in recent years; however, further optimization of engineered PETase toward industrial application has been limited by lower throughput techniques used in protein purification and activity detection. Here, we address these deficiencies through development of a higher-throughput PETase engineering platform. Secretory expression via YebF tagging eliminates lysis and purification steps, facilitating production of large mutant libraries. Fluorescent detection of degradation products permits rapid screening of depolymerase activity in microplates as opposed to serial chromatographic methods. This approach enabled development of more stable PETase, semi-rational (SR) PETase variant containing previously unpublished mutations. SR-PETase releases 1.9-fold more degradation products and has up to 7.4-fold higher activity than wild-type PETase over 10 days at 40°C. These methods can be adapted to a variety of chemical environments, enabling screening of PETase mutants in applications-relevant conditions. Overall, this work promises to facilitate advancements in PETase engineering toward industrial depolymerization of plastic waste.

Directed Immobilization of PETase on Mesoporous Silica Enables Sustained Depolymerase Activity in Synthetic Wastewater Conditions.

Microplastic accumulation in terrestrial and aquatic environments is a growing environmental challenge. Biodegradation has shown promise as an intervention strategy for reducing the spread of microplastics. The wastewater treatment system is a key intervention point in microplastic biodegradation due to its pivotal role in the water cycle at the interface between human activity and the environmental. However, the best characterized microplastic degradation enzyme, PETase, lacks the stability to perform at scale in wastewater treatment. In this work, we show that genetic fusion of PETase to a silica binding peptide enables directed immobilization of the enzyme onto silica nanoparticles. PETase activity in simulated wastewater conditions is quantified by linear regression from time zero to the time of maximum fluorescence of a fluorescent oxidized product of PETase degradation of PET microfibers. Mesoporous silica is shown to be a superior support material to nonporous silica. The resulting biocatalytic nanomaterial has up to 2.5-fold enhanced stability and 6.2-fold increased activity compared to free enzyme in unbuffered, 40 °C simulated influent (ionic strength ∼15 mM). In unbuffered, 40 °C simulated effluent (ionic strength ∼700 µM), reaction velocity and overall catalytic activity were increased by the biocatalytic material 2.1-fold relative to free PETase. All reactions were performed in 0.2 mL volumes, and enzyme concentrations were normalized across both free and immobilized samples to 9 µg/mL. Site-directed mutagenesis is shown to be a complementary technique to directed immobilization, which may aid in optimization of the biomaterial for wastewater applications. PETase stabilization in application-relevant environments as shown here enables progress toward application of PETase for microplastic biodegradation in wastewater treatment.

Engineering Biorthogonal Phage-Based Nanobots for Ultrasensitive, In Situ Bacteria Detection.

Advances in synthetic biology, nanotechnology, and genetic engineering are allowing parallel advances in areas such as drug delivery and rapid diagnostics. Although our current visions of nanobots may be far off, a generation of nanobots synthesized by engineering viruses is approaching. Such tools can be used to solve complex problems where current methods do not meet current demands. Assuring safe drinking water is crucial for minimizing the spread of waterborne illnesses. Although extremely low levels of fecal contamination in drinking water are sufficient to cause a public health risk, it remains challenging to rapidly detect Escherichia coli, the standard fecal indicator organism. Current methods sensitive enough to meet regulatory standards suffer from either prohibitively long incubation times or requirement of expensive, impractical equipment. Bacteriophages, tuned by billions of years of evolution to bind viable bacteria and readily engineered to produce custom proteins, are uniquely suited to bacterial detection. We have developed a biosensor platform based on magnetized phages encoding luminescent reporter enzymes. This system utilizes bio-orthogonally functionalized phages to enable site-specific conjugation to magnetic nanoparticles. The resulting phage-based nanobots, when combined with standard, portable field equipment, allow for detection of <10 cfu/100 mL of viable E. coli within 7 h, faster than any methods published to date.