Abstract
Due to their fast growth rate and robustness, some haloalkalitolerant methanotrophs from the genus Methylotuvimicrobium 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 Methylotuvimicrobium bacteria, a counterselectable marker (CSM) was developed based on pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase. Two-point mutations (T252A and A306G) were introduced into PheS in Methylotuvimicrobium buryatense 5GB1C, generating PheSAG, which can recognize p-chloro-phenylalanine (p-Cl-Phe) as a substrate. Theoretically, the expression of PheSAG in a cell will result in the incorporation of p-Cl-Phe into proteins, leading to cell death. The Ptac promoter and the ribosome-binding site region of mmoX were employed to control pheSAG, producing the pheSAG-3 CSM. M. buryatense 5GB1C harboring pheSAG-3 was extremely sensitive to 0.5 mM p-Cl-Phe. Then, a positive and counterselection cassette, PZ (only 1.5 kb in length), was constructed by combining pheSAG-3 and the zeocin resistance gene. A PZ- and PCR-based strategy was used to create the unmarked deletion of glgA1 or the whole smmo operon in M. buryatense 5GB1C and Methylotuvimicrobium alcaliphilum 20Z. The positive rates were over 92%, and the process could be accomplished in as few as eight days.
Highlights
Methane, the principal component of natural gas and biogas, is a major candidate source of carbon forchemical synthesis (Hanson and Hanson, 1996), and the conversion of methane into valuable products has been pursued off and on for almost half a century (Strong et al, 2015)
Strains 5GB1C and 20Z exhibited similar sensitivity to p-Cl-Phe. The growth of both strains was slightly inhibited in the presence of 0.5 mM p-Cl-Phe, moderately inhibited at 0.8 or 1.0 mM p-Cl-Phe, and greatly inhibited with the addition of 2 mM p-Cl-Phe. These results indicate that the concentration of p-Cl-Phe to be added during counterselection should be less than 1 mM
Strong transcription and translation signals were used to enhance the expression level of PheS with T251A and A294G substitutions (PheSAG) to the extent that the host cell failed to grow in the presence of 0.5 mM p-Cl-Phe, demonstrating that the pheSAG3 expression cassette was an effective counterselectable marker (CSM)
Summary
The principal component of natural gas and biogas, is a major candidate source of carbon for (bio)chemical synthesis (Hanson and Hanson, 1996), and the conversion of methane into valuable products has been pursued off and on for almost half a century (Strong et al, 2015). For strains 5GB1C and 20Z, the genome, transcriptome, and metabolic pathway have been well characterized (Kalyuzhnaya et al, 2013; Strong et al, 2016), the genetic tools have been established (Kalyuzhnaya et al, 2015; Puri et al, 2015; Yan et al, 2016; Nguyen et al, 2018), and metabolic engineering to generate value-added products from methane has been attempted (Andrea et al, 2015; Demidenko et al, 2016; Henard et al, 2016; Nguyen et al, 2018). Promising strains and synthetic biology give new optimism for a realized methane-based bio-industry
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