A promoter–RBS library for fine-tuning gene expression in Methanosarcina acetivorans

ABSTRACT Methanogens are the main biological producers of methane on Earth. Methanosarcina acetivorans is one of the best characterized methanogens that has powerful genetic tools for genome editing. To study the physiology of this methanogen in further detail as well as to effectively balance the flux of their engineered metabolic pathways in expansive project undertakings, there is the need for controlled gene expression, which then requires the availability of well-characterized promoters and ribosome-binding sites (RBS). In this study, we constructed a library of 33 promoter–RBS combinations that includes 13 wild-type and 14 hybrid combinations, as well as six combination variants in which the 5'-untranslated region (5'UTR) was rationally engineered. The expression strength for each combination was calculated by inducing the expression of the β-glucuronidase reporter gene in M. acetivorans cells in the presence of the two most used growth substrates, either methanol (MeOH) or trimethyl amine (TMA). In this study, the constructed library covers a relatively wide range (140-fold) between the weakest and strongest promoter–RBS combination as well as shows a steady increase and allows different levels of gene expression. Effects on the gene expression strength were also assessed by making measurements at three distinct growth phases for all 33 promoter–RBS combinations. Our promoter–RBS library is effective in enabling the fine-tuning of gene expression in M. acetivorans for physiological studies and the design of metabolic engineering projects that, e.g., aim for the biotechnological valorization of one-carbon compounds. IMPORTANCE Methanogenic archaea are potent producers of the greenhouse gas methane and thus contribute substantially to global warming. Under controlled conditions, these microbes can catalyze the production of biogas, which is a renewable fuel, and might help counter global warming and its effects. Engineering the primary metabolism of Methanosarcina acetivorans to render it better and more useful requires controllable gene expression, yet only a few well-characterized promoters and RBSs are presently available. Our study rectifies this situation by providing a library of 33 different promoter–RBS combinations with a 140-fold dynamic range in expression strength. Future metabolic engineering projects can take advantage of this library by using these promoter–RBS combinations as an efficient and tunable gene expression system for M. acetivorans. Furthermore, the methodologies we developed in this study could also be utilized to construct promoter libraries for other types of methanogens.

b For promoter/RBS-hybrid combinations: the exchanged RBS region (RBSmcr) of the wild-type promoter-RBS combination via V1 strategy (see Figure 2A) is marked with wave underline, and the exchanged promoter region via the V2 strategy is underlined.
c For the 5'UTR-engineered combinations: mutated (enlarged uppercase boldface font) and deleted (strikethrough boldface font) bases are indicated.

Figure S2 .
Figure S2.Predicted RNA secondary structures of the entire 5'UTR sequence in the promoter-RBS combination of PmcrB_mm and its six variants.RNA secondary structures and their free energies were predicted with NUPACK (https://www.nupack.org/)using default settings.All 5'UTRs are in the 5'-to-3' direction.Bases in the RNA sequence are designated as colored dots (A, green; C, blue; G, black; and U, red).DNA sequences for the promoter (PmcrB_mm from M. mazei) and consensus RBS motif (GGAGG) are underlined.Promoter-RBS combination variants (PUTR1, PUTR2, PUTR3, PUTR4, PUTR5, and PUTR6) are derived from PmcrB_mm.Mutation sites in each variant are annotated in the secondary structure.The location of the start codon (AUG) is shown.

Figure S3 .
Figure S3.Growth curves of various promoter-RBS combination strains in MeOH.For comparative purposes, all data points represent an average value of three replicates.(A) Strains expressing strong promoter-RBS combinations.(B) Strains expressing medium promoter-RBS combinations.(C) Strains expressing weak promoter-RBS combinations.An empty vector strain (PZ0 strain containing the uidA cassette but lacking the promoter-RBS sequence) is the control (dark gray square).All cultures reached an OD600 > 1.7 after a 40-hour incubation period (OD600 = 1.77 for the control).

Figure
Figure S4.(A) Sequence alignment of hdrED1 promoter-RBSs from different Methanosarcina species.Promoter sequences are derived as follows: PhdrE_mm from M. mazei, PhdrE_ma from M. acetivorans, and PhdrE_mb from M. barkeri.A multiple alignment of DNA sequences was performed with MultAlin (http://multalin.toulouse.inra.fr/multalin/multalin.html)using default settings.Highly conserved regions (> 90% similarity) are indicated (red font).Sequences for the putative promoter elements are indicated: BRE (dashed line bordering), TATA box (solid line bordering), and TSS (black arrow).The putative RBS sequence is shown (purple).The start codon (ATG) is underlined in each DNA sequence.(B to D) Prediction of 5'UTR secondary structures and their free energies.RNA secondary structures and their free energies were predicted with NUPACK (https://www.nupack.org/)using default settings.All 5'UTRs are in the 5'-to-3' direction.Bases in the RNA sequence are designated as colored dots (A, green; C, blue; G, black; and U, red).The location of the putative RBS is indicated by a solid line.The location of the start codon (AUG) is shown.

Figure S5 .
Figure S5.Standard curve of E. coli β-glucuronidase activity.Activity measurements were performed according to the assay protocol described in Materials and Methods (see main text).

Table S3 . Strains and plasmids used in this study.
a The plasmid sequence is available by clicking the link.