Development of Enhanced Transformation and Promoters to Engineer an Environmental-Isolate of Priestia Megaterium.

Engineering microbes for sustainable bioproduction is a promising avenue to tackle problems in pollution, medicine, and energy. Traditional model organisms have received much attention due to their easily manipulated genetics and well characterized growth. However, there are a wealth of non-model microbes with attributes that are beneficial for biotechnological applications, but genetic tools must first be developed to engineer them by incorporating foreign DNA and characterizing promoters for controlled expression. In this work, we are looking to expand the genetic toolbox for an environmental-isolate of Priestia megaterium, called SR7, that can survive under high pressures of CO₂, simultaneously creating a sterile culture environment as well as extracting bioproducts as they are formed. For these reasons, SR7 was previously engineered to produce isobutanol, a second-generation biofuel, under supercritical CO₂.

Genetic manipulation of SR7 was previously possible using a protoplast-osmotic shock method, but was highly inefficient, necessitating the development of an enhanced transformation method. Plasmid methylation was controlled using a strain of E. coli which lacks native methylation (dam, dcm mcrA, mcrC) genes and contains a methyltransferase gene from SR7. Using the methylation strain as well as osmotic balancing agents, transformation efficiencies have been enhanced, permitting bioparts validation. We next sought to apply constitutive promoters to our bioproduction pathway by mining a transcriptome of SR7 grown in different environments. Several promoters were identified and characterized, finding particularly high expression from the native citrate synthase promoter. This has allowed us to begin experimentally testing gene expression levels which will lead to an optimized set of bioparts to produce isobutanol.