The rapid increase of antibiotic-resistant bacteria poses serious global health concerns. Doctors struggle to treat multi-drug-resistant bacterial infections, which can be caused by dangerous pathogens like Carbapenem-resistant Acinetobacter baumannii, Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Enterococcus Sp. (VRE), Carbapenem-resistant Pseudomonas aeruginosa, and multi-drug-resistant Mycobacterium tuberculosis (MDR-TB). Each year, these pathogens are responsible for millions of deaths globally. Today, there is an urgent need for the development of new antibiotic agents to combat multi-drug resistant pathogens. Unfortunately, creating effective antibiotics has been time-consuming and costly. 

A promising antimicrobial peptide called “Thanatin,” produced by the insect called spined soldier bug (Podisus meculiventris), has been proven to have a wide range of antimicrobial activity. Thanatin accomplishes this by binding to LPS in the bacterial cell wall, inhibiting its biosynthesis, and permeabilizing bacterial cell membranes. The combination of these activities can kill many types of bacterial pathogens.

For our project, we aim to engineer yeast Pichira pastoris to produce Thanatin for treating multi-drug-resistant bacteria. We intend to use P. pastoris as our chassis because it is widely used in bioengineering and can efficiently and cost-effectively produce large amounts of recombinant Thanatin. To accomplish this, we will clone the DNA sequences that encode the Thanatin-GFP fusion peptide into yeast expression vector pGAP4_r, marked by Zeo for selection. This construct will then be transformed into P. pastoris. 

Next, we will use a fluorescence microscope to visualize the green fluorescent protein (GFP) that is part of the Thanatin-GFP fusion peptide to confirm protein production. To test Thanatin’s antimicrobial properties, we will purify it and perform MIC testing to determine its ability to kill pathogenic and MDR bacteria.