(70d) Developing Genetic Engineering Tools and Adaptive Laboratory Evolution for Priestia Megaterium SR7 Under High CO2 Conditions

Over the last century, the increasing dependence on non-renewable fossil sources has driven the demand and motivation to convert renewable feedstocks into valuable products. Recent developments in biotechnology have provided more options for producing these products by utilizing sustainable materials. However, the high production costs of bio-based products, largely due to intricate downstream processes, and the predominance of batch production in bioprocessing, to reduce contamination risks, pose significant challenges. To address these issues, we are investigating the use of Priestia megaterium SR7 as a platform for biochemical production under high CO₂ conditions, including supercritical CO₂ (scCO₂).

As a first step, we developed and optimized a suite of genetic engineering tools specifically for P. megaterium SR7, including electroporation for plasmid delivery, CRISPR-Cas9, dead Cas9 (dCas9), and adenine base editors. These tools are being employed to study the strain's physiology and to engineer improved phenotypes. In parallel, we are conducting adaptive laboratory evolution (ALE) experiments to enhance the strain's tolerance to high CO₂ conditions. Currently, we have evolved P. megaterium SR7 under 100% CO₂ at atmospheric pressure (1 atm). Multi-omics analysis was employed to analyze the changes throughout the passages. Genome sequencing of the evolved strain revealed a deletion of over 20 kilobase pairs, the loss of a native plasmid, and more than 30 nonsynonymous mutations. These results were validated by introducing the changes to the wildtype strain using the developed genetic engineering tools.

To further investigate the phenotype of the evolved strain, we creating a dCas9 genome-wide library and culturing the libraries derived from the evolved strain under 100% CO₂ at 1 atm for 10 generations. By comparing the increased and decreased libraries, we aim to identify candidate genes associated with a positive effect on the strain's growth.

Ongoing work aims to further evolve P. megaterium SR7 to tolerate increasingly higher pressures of CO₂, with the ultimate goal of reaching scCO₂ conditions. We are also exploring the activation of a cryptic pathway, such as the 2,3-butanediol (2,3-BDO) pathway, in the evolved strains to produce value-added chemicals. By combining genetic engineering, ALE, and multi-omics analyses, we aim to develop a robust and sustainable platform for biochemical production under high CO₂ conditions, potentially enabling more efficient and economical downstream processing through the use of scCO₂ extraction methods.