Metabolic engineering of a novel strain of electrogenic bacterium Arcobacter butzleri to create a platform for single analyte detection using a microbial fuel cell

• Published in: Enzyme and microbial technology 2020-09, Vol.139, p.109564-109564, Article 109564
• Main Authors: Szydlowski, Lukasz, Lan, Tammy C.T., Shibata, Noriko, Goryanin, Igor
• Format: Article
• Language: English
• Subjects: Biosensor; CRISPR; Electrode associated bacteria; Electron transfer; Microbial fuel cell
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2020.109564

[Display omitted] Highlights •We demonstrate that it is possible to engineer the genome of an electrogenic bacterium using CRISPR system, with either Cas9 or Cpf1 and using RNP complex delivery.•We have demonstrated successful genome editing of a novel strain of epsilon proteobacterium enriched on the anode of microbial fuel cells (MFC); knockout mutants were limited to grow on either acetate or lactate as their carbon source.•Albeit metabolically limited, these mutants were still able to produce electricity, but current generation was limited to a single carbon source that these mutants were able to metabolize Electrogenic bacteria metabolize organic substrates by transferring electrons to the external electrode, with subsequent electricity generation. In this proof-of-concept study, we present a novel strain of a known, electrogenic Arcobacter butzleri that can grow primarily on acetate and lactate and its electric current density is positively correlated (R2 = 0.95) to the COD concentrations up to 200 ppm. Using CRISPR-Cas9 and Cpf1, we engineered knockout Arcobacter butzleri mutants in either the acetate or lactate metabolic pathway, limiting their energy metabolism to a single carbon source. After genome editing, the expression of either acetate kinase, ackA, or lactate permease, lctP, was inhibited, as indicated by qPCR results. All mutants retain electrogenic activity when inoculated into a microbial fuel cell, yielding average current densities of 81–82 mA/m2, with wild type controls reaching 85–87 mA2. In the case of mutants, however, current is only generated in the presence of the substrate for the remaining pathway. Thus, we demonstrate that it is possible to obtain electric signal corresponding to the specific organic compound via genome editing. The outcome of this study also indicates that the application of electrogenic bacteria can be expanded by genome engineering.