pheS AG Based Rapid and Efficient Markerless Mutagenesis in Methylotuvimicrobium

Due to their fast growth rate and robustness, some haloalkalitolerant methanotrophs from the genus have recently become not only promising biocatalysts for methane conversion but also favorable materials for obtaining fundamental knowledge on methanotrophs. Here, to realize unmarked genome modification in bacteria, a counterselectable marker (CSM) was developed based on , which encodes the α-subunit of phenylalanyl-tRNA synthetase. Two-point mutations (T252A and A306G) were introduced into PheS in 5GB1C, generating PheS , which can recognize -chloro-phenylalanine ( -Cl-Phe) as a substrate. Theoretically, the expression of PheS in a cell will result in the incorporation of -Cl-Phe into proteins, leading to cell death. The P promoter and the ribosome-binding site region of were employed to control , producing the CSM. 5GB1C harboring was extremely sensitive to 0.5 mM -Cl-Phe. Then, a positive and counterselection cassette, PZ (only 1.5 kb in length), was constructed by combining and the zeocin resistance gene. A PZ- and PCR-based strategy was used to create the unmarked deletion of or the whole operon in 5GB1C and 20Z. The positive rates were over 92%, and the process could be accomplished in as few as eight days.