(265f) Engineering the Cyanobacterium Synechococcus sp. PCC 11901 for D-Lactate Bioproduction Using MEPS.

Cyanobacteria, gram-negative prokaryotic microorganisms, offer substantial potential in the bioindustry as sustainable and efficient platforms for biofuels and biochemical production. Among these, Synechococcus sp. PCC 11901 is notable as a fast-growing marine cyanobacterium that is naturally competent and compatible with synthetic biology tools used in engineering Escherichia coli, making it highly manipulable compared to other photoautotrophs such as plants and algae. This study focuses on engineering Synechococcus sp. PCC 11901 for D-lactate production through metabolic engineering and synthetic biology approaches in a Microbial Electrosynthesis (MEPS) reactor, facilitated by an electron mediator. This endeavor aims to enable economical and sustainable production of D-lactate.

The engineering of cyanobacteria for D-lactate production presents unique challenges due to their polyploidy and slow growth rate. However, advancements in CRISPR-based genome engineering, including the use of non-toxic Cas9 alternatives like cpf1 from Francisella novicida, will significantly reduce segregation times. Additionally, multiplex genome editing strategies are employed to streamline gene integration processes. The adaptation of 11901 strains with recJ knockout will enhance the transformation efficiency, while different high-level expression of gRNAs facilitates efficient gene knockouts.Notably, the PCC 11901 genome contains two recJ genes, underscoring the importance of identifying the specific recJ responsible for the improved efficiency. To address this, individual constructs targeting each recJ gene separately were designed, and their effects on transformation efficiency were compared. Furthermore, two plasmids were constructed for recJ deletion using homologous recombination to insert cpf1.

A critical step in photosynthesis is carried out by Photosystem II (PSII), which uses light energy to split water into protons, electrons and O₂; however, PSII is damaged under even modest light intensities and thereby limits electron flow. To enable Synechococcus 11901 on MEPS, PSII will be inactivated by creating a psbB mutant (ΔpsbB) using CRISPR. The mutant strains were leveraged to integrate the D-lactate gene(GlyDH*) by knocking out the psbB gene responsible for encoding chlorophyll protein, with the pUC18 vector serving as the backbone for these constructs. The D-lactate strain will be grown in the MEPS reactor in the presence of an electron mediator and D-lactate production will be monitored by varying light and CO₂.