Investigating Streptomyces clavuligerus Linear Replicons for Improved Clavulanic Acid Production

Increasing antimicrobial resistance against beta-lactam antibiotics through bacterially produced beta-lactamases has prompted research into effective enzyme inhibitors, such as clavulanic acid. Streptomyces clavuligerus (Sclav) is the primary producer of clavulanic acid, which is used in various drugs such as Augmentin(r). Sclav has four giant linear plasmids (GLPs) in addition to its chromosome and produces linear replicons with conserved, palindromic telomeric sequences. This project will focus on investigating plasmid-chromosome interactions with the aim of ultimately curing GLPs to decrease the metabolic burden and increase clavulanic acid production.
In order to investigate the role of the GLPs and their interaction with the chromosome, the significance of terminal proteins, Tap and Tpg, involved in telomeric replication, and the importance of their presence on the GLPs, will be determined. This will be done by constructing a CRIPSR-dcas9 knockdown of tap and tpg.
The absence of Tap and Tpg from the GLPs is expected to have an impact on chromosomal linearity, as chromosomal shortening has previously led to circularisation. To overcome chromosomal shortening, tap and tpg will be incorporated in the chromosome, aiding potential GLP curing. The lack of endonuclease activity in CRISPR-dcas9 is expected decrease the interference with more sensitive Sclav strains, allowing for increased strain stability.
Telomere replication depends on the interaction of palindromic sequences with Tap and Tpg terminal proteins encoded on three of the plasmids. Understanding the complex genome architecture of Sclav is vital to genetically engineering strains with increased fermenter stability and clavulanic acid productivity.