Lactacystin Biosynthesis

Project Information

Principal Investigator: Yousong Ding
Institution: University of Florida
College: Pharmacy
Department: Medicinal Chemistry
Time Affiliated with Project: Spring 2023- Present

Project Background

Streptomyces lacticystinicus (S. lac) is a gram-positive bacteria strain that produces lactacystin, a nature product that acts as a proteosome inhibitor. As a proteosome inhibitor, lactacystin has the capacity to regulate many metabolic processes such as the cell cycle, apoptosis, gene expression, and DNA repair. These bioactivities make lactacystin an attractive drug lead for multiple myeloma, gastric cancer, malaria, and many other notable diseases.

Exploring Lactacystin Biosynthesis

The aim of this project is to identify, clone, and express the lactacystin biosynthetic gene cluster (BGC) by using genome mining and heterologous expression. The initial phases of the project primarily rely on genome mining via bioinformatics. Programs like ANTISMASH allow us to compare domains of S.lac genomic DNA with bacteria that produce lactacystin analogues to hypothesize the lactacystin BGC. The proposed BGC allows us to design experimental conditions as we prepare for heterologous expression. Our current cloning strategy consists of isolating the S. lac genomic DNA, isolating the BGC by PCR, assembling the BGC into a vector, and cloning the BGC within Escherichia coli DH5α. Upon successful cloning, we plan to do heterologous expression in various streptomyces species. Heterologous expression will confirm that the BGC was properly identified, and an effective cloning strategy was developed.

Throughout the project it is important to check the results of each experimental step. The following images are from gel electrophoresis, which separates DNA based on the size of the fragments. A ladder serves as a reference so we can check experimental band size with the expected band size.

Pictured above are BGC fragments that were amplified via PCR. The PCR bands appear strongly at the expected regions.

Pictured above is the digested plasmid that will serve as the vector when assembling the BGC. The topmost band corresponds to the correctly digested plasmid.

My Research Interests

Natural product discovery

Biosynthesis

Pharmaceutical Clinical Trials

Total Organic Synthesis

My Role

I have been lucky enough to work on this project for an extended period and my responsibilities have changed as I have learned more about the project. In spring 2023, I primarily learned the experimental techniques for each phase of the project. During this time, I was supervised while learning protocols for heat shock transformation, PCR, digestion, and DNA/plasmid purification. I took on a larger role in the project in spring 2024. I participated in the project planning by reading papers that studied similar biosynthetic pathways and studying the lactacystin BGC active domains on ANTISMASH. In summer 2024, I began working on independently performing the steps for the cloning and assembly phase of the project.