Abstract
Polyamines are metabolites essential for cellular metabolism and functions, but their accumulation can become cytotoxic and contribute to diseases, such as cancer. This highlights the need for strategies to regulate polyamine levels. To address this, surface expression of BltD, a polyamine acetyltransferase from Bacillus subtilis, was explored to enable direct acetylation of extracellular polyamines and support polyamine homeostasis. BltD was surface-displayed in Escherichia coli UT5600 and UT2300 through the BrkA autotransporter. In silico structural modelling supported proper folding and compatibility of the BrkA-BltD fusion. A recombinant plasmid, pBOTH, was constructed using Gibson assembly by inserting bltD into the BrkA passenger domain, with a hexahistidine (6x His) tag for detection and an OmpT cleavage site for potential extracellular processing. Plasmid construction was validated by colony PCR and Nanopore sequencing. Following the transformation of pBOTH into UT5600 and UT2300, trypsin accessibility and Western blot analysis showed loss of BrkA-BltD bands after trypsin treatment, suggesting surface localization of BltD in both strains. Growth assays demonstrated that pBOTH expression did not impair growth in either strain. Together, these findings demonstrated that BltD was surface-displayed via the BrkA autotransporter in both UT5600 and UT2300, establishing a potential platform for the extracellular acetylation of polyamines and future investigation of polyamine regulation.