Enhancing genetic engineering of Methanococcus maripaludis by evaluating integration sites and testing ferredoxin replaceability in homocysteine biosynthesis

Abstract

Methanococcus maripaludis is a genetically tractable and fast-growing methanogen that is attractive for both a rchaeal biology studies and biotechnological applications involving conversion of CO2 and H2 to biofuels and other valuable biochemicals. However, lack of integration sites and profound comprehension on ferredoxin interchangeability hinders its more complex genetic engineering.

Integration of heterologous genes or pathways into the host’s genome is critical for stable and long-term expression important in both biological and biotechnological studies. In order to enhance the biotechnological potential of M. maripaludis, gene expression of seven integration sites was analyzed using a β-glucuronidase activity assay. The β-glucuronidase gene was inserted into the integration sites with a CRISPR-Cas12a-based genome editing toolbox. All integration sites showed detectable β-glucuronidase activity.

In M. maripaludis, ferredoxins transfer electrons in metabolically important pathways such as methanogenesis, which is reflected by their high variety. Ferredoxin pools in M. maripaludis are rather complex and their interchangeability is not fully understood. From the perspective of genetic engineering, understanding ferredoxin interchangeability becomes crucial for both the function of heterologous enzymes that require ferredoxins as electron carriers, and re-estabilishing the redox balance after introducing heterologous pathways.

Homocysteine biosynthesis is one of the many ferredoxin-associated reactions in M. maripaludis and used in this study as a test case. Homocysteine is synthetized from aspartate semialdehyde and hydrogen sulfide via MMJJ_14800/MMJJ_14810 -dependent pathway, where MMJJ_14800 is an enzyme and MMJJ_14810 a ferredoxin. To investigate the role of MMJJ_14810 in the reaction, phenotypic assays of the knock-out strains ΔMMJJ_14800 and ΔMMJJ_14810 were conducted in minimal medium with and without homocysteine. In minimal medium, strain ΔMMJJ_14810 demonstrated impaired growth , indicating that ferredoxin MMJJ_14810 is interchanged, yet imperfectly. This study facilitates genetic engineering of M. maripaludis by providing valuable information on integration sites and ferredoxin interchangeability.