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.

Keywords: 5'UTR-engineering; Methanosarcina acetivorans; RBS; gene expression; library construction; methanogens; promoter.

Fig 1

Characterization of wild-type promoter–RBS combinations from different methanogens. (A) Scheme for integration of the promoter–RBS–uidA cassette fusion into the M. acetivorans genome. The uidA cassette comprises the uidA gene from E. coli BL21 and the mcr terminator (T_mcr) from M. barkeri. (B) Expression strength of the eleven wild-type promoter–RBS combinations (black bar) during growth on MeOH. The minimal P_mcrB promoter (orange bar) was used as a control for performing strength comparison. Error bars represent the standard deviation of three biological replicates.

Fig 2

Strategies used to obtain medium-strength promoter–RBS combinations. Strategy 1. (A) Construction of promoter–RBS/hybrid variants (see the main text for details). Two variants (V1 and V2) are derived from a weak wild-type promoter–RBS combination (in blue). V1 possesses the weak wild-type promoter and the strong RBS_mcr from P_{mcrB_mb}. V2 possesses the strong P_{mcrB_mb} promoter and the weak wild-type RBS. P_{mcrB_mb} is the strong wild-type promoter–RBS combination from M. barkeri. (B) Expression strength comparison of seven wild-type promoter–RBS combinations (orange font and light gray bar) and their promoter–RBS/hybrid variants. Variants of V1 (dark gray bar) and V2 (black bar) are denoted as −1 and −2, respectively. Error bars represent the standard deviation of three biological replicates (*P < 0.05, **P < 0.01, and ***P < 0.001). Strategy 2. (C) Expression strength of the wild-type P_atpH promoter–RBS combinations from the Methanosarcina species. Promoters are derived as follows: P_{atpH_mm} from M. mazei, P_{atpH_mb} from M. barkeri, and P_{atpH_ma} from M. acetivorans. The minimal P_mcrB (orange bar) promoter was used for making expression strength comparison. (D) Predicted (partial) 5'UTR secondary structure of P_{atpH_ma}, P_{atpH_mb} (left), and P_{atpH_mm} (right). Secondary structures and their free energies were predicted with NUPACK (https://www.nupack.org/) using default settings. Loops 1 and 2 are the predicted secondary structures in the extra 57-bp sequence of the 5'UTR in P_{atpH_mm}. This additional sequence is not present in the 5'UTR of P_{atpH_ma} and P_{atpH_mb}. The start codon (AUG) in the mRNA sequence is indicated.

Fig 3

Engineered promoter–RBS combination variants with stronger expression than P_{mcrB_mm}. (A) RNA secondary structure predictions of the last 30-bp sequence in the P_{mcrB_mm} 5'UTR for six promoter–RBS combination variants (P_UTR1, P_UTR2, P_UTR3, P_UTR4, P_UTR5, and P_UTR6). Mutation sites in each variant are annotated in the secondary structure. RNA secondary structures were predicted with NUPACK using default settings. RNA secondary structures for the full-length 5'UTR sequence of the six promoter–RBS combination variants as well as their free energies are provided in Fig. S2. The consensus RBS motif sequence (5´-GGAGG) in the Methanosarcina species and the start codon (AUG) in the mRNA sequence are both indicated. (B) Expression strengths of the P_{mcrB_mm} variant strains (black bar) during growth on MeOH. Strength comparisons included using wild-type P_{mcrB_mm} (beige bar) and minimal P_mcrB (orange bar) as controls. Error bars represent the standard deviation of three biological replicates.

Fig 4

Expression strength of all 33 promoter–RBS combinations grown on MeOH or TMA as the sole carbon source. Growth on MeOH (black bar) or TMA (brass bar) is indicated. The minimal P_mcrB promoter (orange font) was used as the reference to categorize the promoter–RBS combinations as either strong (> 100%), medium (20%–100%), or weak (< 20%) (dashed line). Error bars represent the standard deviation of three biological replicates (**P < 0.01 and ***P < 0.001).

Fig 5

Expression strength of all 33 promoter–RBS combinations during different growth phases. MeOH was used as the sole carbon and energy source. Phases of cell growth are marked as follows: early exponential phase (OD600 = 0.3–0.5) (dark orange bar), mid-exponential phase (OD600 = 0.5–0.8) (orange bar), and late exponential–early stationary phase (OD600 = 0.8–1.2) (light orange bar). Promoter–RBS combinations showing increased gene expression and cell growth are indicated by black arrows. Error bars represent the standard deviation of three biological replicates.