Efficient in planta production of amidated antimicrobial peptides that are active against drug-resistant ESKAPE pathogens.

Antimicrobial peptides (AMPs) are promising next-generation antibiotics that can be used to combat drug-resistant pathogens. However, the high cost involved in AMP synthesis and their short plasma half-life render their clinical translation a challenge. To address these shortcomings, we report efficient production of bioactive amidated AMPs by transient expression of glycine-extended AMPs in Nicotiana benthamiana line expressing the mammalian enzyme peptidylglycine α-amidating mono-oxygenase (PAM). Cationic AMPs accumulate to substantial levels in PAM transgenic plants compare to nontransgenic N. benthamiana. Moreover, AMPs purified from plants exhibit robust killing activity against six highly virulent and antibiotic resistant ESKAPE pathogens, prevent their biofilm formation, analogous to their synthetic counterparts and synergize with antibiotics. We also perform a base case techno-economic analysis of our platform, demonstrating the potential economic advantages and scalability for industrial use. Taken together, our experimental data and techno-economic analysis demonstrate the potential use of plant chassis for large-scale production of clinical-grade AMPs.

Microbial Biocontainment Systems for Clinical, Agricultural, and Industrial Applications.

Many applications of synthetic biology require biological systems in engineered microbes to be delivered into diverse environments, such as for in situ bioremediation, biosensing, and applications in medicine and agriculture. To avoid harming the target system (whether that is a farm field or the human gut), such applications require microbial biocontainment systems (MBSs) that inhibit the proliferation of engineered microbes. In the past decade, diverse molecular strategies have been implemented to develop MBSs that tightly control the proliferation of engineered microbes; this has enabled medical, industrial, and agricultural applications in which biological processes can be executed in situ. The customization of MBSs also facilitate the integration of sensing modules for which different compounds can be produced and delivered upon changes in environmental conditions. These achievements have accelerated the generation of novel microbial systems capable of responding to external stimuli with limited interference from the environment. In this review, we provide an overview of the current approaches used for MBSs, with a specific focus on applications that have an immediate impact on multiple fields.

CS-Cells: A CRISPR-Cas12 DNA Device to Generate Chromosome-Shredded Cells for Efficient and Safe Molecular Biomanufacturing.

Synthetic biology holds great promise for translating ideas into products to address the grand challenges facing humanity. Molecular biomanufacturing is an emerging technology that facilitates the production of key products of value, including therapeutics and select chemical compounds. Current biomanufacturing technologies require improvements to overcome limiting factors, including efficient production, cost, and safe release; therefore, developing optimum chassis for biomolecular manufacturing is of great interest for enabling diverse synthetic biology applications. Here, we harnessed the power of the CRISPR-Cas12 system to design, build, and test a DNA device for genome shredding, which fragments the native genome to enable the conversion of bacterial cells into nonreplicative, biosynthetically active, and programmable molecular biomanufacturing chassis. As a proof of concept, we demonstrated the efficient production of green fluorescent protein and violacein, an antimicrobial and antitumorigenic compound. Our CRISPR-Cas12-based chromosome-shredder DNA device has built-in biocontainment features providing a roadmap for the conversion of any bacterial cell into a chromosome-shredded chassis amenable to high-efficiency molecular biomanufacturing, thereby enabling exciting and diverse biotechnological applications.