Construction of a broad-host-range Anderson promoter series and particulate methane monooxygenase promoter variants expand the methanotroph genetic toolbox

Abstract

Methanotrophic bacteria are currently used industrially for the bioconversion of methane-rich natural gas and anaerobic digestion-derived biogas to valuable products. These bacteria may also serve to mitigate the negative effects of climate change by capturing atmospheric greenhouse gases. Several genetic tools have previously been developed for genetic and metabolic engineering of methanotrophs. However, the available tools for use in methanotrophs are significantly underdeveloped compared to many other industrially relevant bacteria, which hinders genetic and metabolic engineering of these biocatalysts. As such, expansion of the methanotroph genetic toolbox is needed to further our understanding of methanotrophy and develop biotechnologies that leverage these unique microbes for mitigation and conversion of methane to valuable products. Here, we determined the copy number of three broad-host-range plasmids in Methylococcus capsulatus Bath and Methylosinus trichosporium OB3b, representing phylogenetically diverse Gammaproteobacterial and Alphaproteobacterial methanotrophs, respectively. Further, we show that the commonly used synthetic Anderson series promoters are functional and exhibit similar relative activity in M. capsulatus and M. trichosporium OB3b, but the synthetic series had limited range. Thus, we mutagenized the native M. capsulatus particulate methane monooxygenase promoter and identified variants with activity that expand the activity range of synthetic, constitutive promoters functional not only in M. capsulatus, but also in Escherichia coli. Collectively, the tools developed here advance the methanotroph genetic engineering toolbox and represent additional synthetic genetic parts that may have broad applicability in Pseudomonadota bacteria.

Keywords: Metabolic engineering; Methane monooxygenase; Methanotroph; Promoter; Synthetic biology.

Fig. 1

Broad-host-range plasmid copy number in phylogenetically diverse methanotrophs. A) Broad-host-range IncP- (pCAH01), IncQ- (pQCH), and pBBR-based (pBMTL-2) plasmid maps. Replicon and antibiotic resistance genes are highlighted in grey or orange, respectively. B) The cycle threshold (Ct) difference between the single copy RNA polymerase β subunit rpoB gene and the plasmid kanamycin resistance ahp gene in genomic DNA extracted from M. capsulatus or M. trichosporium plasmid-harboring transformants determined by quantitative PCR. C) Plasmid copy number calculated using qPCR data. The data in B and C represent the mean ± SEM from six individual transformants.

Fig. 2

Comparison of the Anderson series promoter activity in E. coli and diverse methanotrophs. Relative Anderson series promoter activity in E. coli (white bar), M. capsulatus (red bar), and M. trichosporium (grey bar) determined by mRFP1 fluorescence during logarithmic growth phase cells. Linear regression analysis comparing relative Anderson series promoter activity in E. coli to that in M. capsulatus (B) or M. trichosporium (C). The data represent the mean ± SEM from two independent experiments (n = 4).

Fig. 3

Particulate methane monooxygenase promoter variant activity in E. coli. A and B) Relative sfGFP fluorescence of selected particulate methane monooxygenase promoter (P_pmoc2) mutagenesis library E. coli transformants harboring a P_pmoc2-sfGFP reporter plasmid. C) Sequence alignment of P_pmoc2 promoter variants with mutations in the core upstream (UP), −35, and −10 promoter elements with measured “low”, “medium”, and “high” activities.

Fig. 4

Particulate methane monooxygenase promoter variants expand the methanotroph genetic toolbox. A) Comparison of mutant and wild-type particulate methane monooxygenase promoter (P_pmoC2) activity in E. coli (white bar) and M. capsulatus (green bar). B) The dynamic range of selected Anderson series and P_pmoC2 variant promoter activity in M. capsulatus.

Fig. S1

Particulate methane monooxygenase promoter variant sequences. Sequence alignment of the particulate methane monooxygenase promoter (P_pmoc2) region in plasmid DNA isolated from twenty-four transformants (P_1-24) obtained after promoter mutagenesis as described in materials and methods and shown in Fig. 3. The upstream (UP), −35, and −10 promoter elements are marked on the wild-type sequence.