(746a) Development of New Reagents and Procedures to Facilitate Cyanobacterial Genetic Engineering

Cyanobacteria and other photosynthetic organisms have tremendous potential as host strains for industrial chemical production driven by energy from the sun. Two important engineering tasks - transformation with exogenous DNA, and measurement of intracellular samples - are difficult in cyanobacteria because they have particularly strong outer peptidoglycan and exopolysaccharide layers that impede DNA access and cell lysis. Currently, large amounts of exogenous DNA are required for reliable transformation of cyanobacteria, which makes it cumbersome to, for example, screen a large library of constructs. Cells must be lysed by disintegration with beads or treatment with harsh solvents, which could damage important cellular components and severely limits the throughput of intracellular measurements.

We are developing a series of novel lysozymes as tools to make both of these tasks easier. Lysozymes, which hydrolyze peptidoglycan chains, could be used to soften the cell wall to improve transformation efficiency and, in a stronger treatment, completely degrade the cell wall to gently lyse cells. Originally discovered in a cyanobacterial community, our lysozymes appears to be particularly suited to treating cyanobacteria. We have used in vivo and cell-free platforms to optimize expression conditions for these enzymes, and have developed procedures for cell lysis and pretreatment during transformation. These engineered enzymes together with the new procedures should serve as valuable tools for the cyanobacterial research and engineering community to improve transformation efficiency and enable biologically gentle, high-throughput cell lysis for intracellular measurement.