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

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 ß-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.

Structure-Assisted Design of Chitosanase Product Specificity for the Production of High-Degree Polymerization Chitooligosaccharides.

Chitosanases are valuable enzymatic tools in the food industry for converting chitosan into functional chitooligosaccharides (COSs). However, most of the chitosanases extensively characterized produced a low degree of polymerization (DP) COSs (DP = 1-3, LdpCOSs), indicating an imperative for enhancements in the product specificity for the high DP COS (DP >3, HdpCOSs) production. In this study, a chitosanase from Methanosarcina sp. 1.H.T.1A.1 (OUC-CsnA4) was cloned and expressed. Analysis of the enzyme-substrate interactions and the subsite architecture of the OUC-CsnA4 indicated that a Ser49 mutation could modify its interaction pattern with the substrate, potentially enhancing product specificity for producing HdpCOSs. Site-directed mutagenesis provided evidence that the S49I and S49P mutations in OUC-CsnA4 enabled the production of up to 24 and 26% of (GlcN)5 from chitosan, respectively─the wild-type enzyme was unable to produce detectable levels of (GlcN)5. These mutations also altered substrate binding preferences, favoring the binding of longer-chain COSs (DP >5) and enhancing (GlcN)5 production. Furthermore, molecular dynamics simulations and molecular docking studies underscored the significance of +2 subsite interactions in determining the (GlcN)4 and (GlcN)5 product specificity. These findings revealed that the positioning and interactions of the reducing end of the substrate within the catalytic cleft are crucial factors influencing the product specificity of chitosanase.

Physiological and transcriptomic response to methyl-coenzyme M reductase limitation in Methanosarcina acetivorans.

Methyl-coenzyme M reductase (MCR) catalyzes the final step of methanogenesis, the microbial metabolism responsible for nearly all biological methane emissions to the atmosphere. Decades of biochemical and structural research studies have generated detailed insights into MCR function in vitro, yet very little is known about the interplay between MCR and methanogen physiology. For instance, while it is routinely stated that MCR catalyzes the rate-limiting step of methanogenesis, this has not been categorically tested. In this study, to gain a more direct understanding of MCR's control on the growth of Methanosarcina acetivorans, we generate a strain with an inducible mcr operon on the chromosome, allowing for careful control of MCR expression. We show that MCR is not growth rate-limiting in substrate-replete batch cultures. However, through careful titration of MCR expression, growth-limiting state(s) can be obtained. Transcriptomic analysis of M. acetivorans experiencing MCR limitation reveals a global response with hundreds of differentially expressed genes across diverse functional categories. Notably, MCR limitation leads to strong induction of methylsulfide methyltransferases, likely due to insufficient recycling of metabolic intermediates. In addition, the mcr operon is not transcriptionally regulated, i.e., it is constitutively expressed, suggesting that the overabundance of MCR might be beneficial when cells experience nutrient limitation or stressful conditions. Altogether, we show that there is a wide range of cellular MCR concentrations that can sustain optimal growth, suggesting that other factors such as anabolic reactions might be rate-limiting for methanogenic growth. IMPORTANCE: Methane is a potent greenhouse gas that has contributed to ca. 25% of global warming in the post-industrial era. Atmospheric methane is primarily of biogenic origin, mostly produced by microorganisms called methanogens. Methyl-coenzyme M reductase (MCR) catalyzes methane formatio in methanogens. Even though MCR comprises ca. 10% of the cellular proteome, it is hypothesized to be growth-limiting during methanogenesis. In this study, we show that Methanosarcina acetivorans cells grown in substrate-replicate batch cultures produce more MCR than its cellular demand for optimal growth. The tools outlined in this study can be used to refine metabolic models of methanogenesis and assay lesions in MCR in a higher-throughput manner than isolation and biochemical characterization of pure protein.

Catalytic Activity of the Archetype from Group 4 of the FTR-like Ferredoxin:Thioredoxin Reductase Family Is Regulated by Unique S = 7/2 and S = 1/2 [4Fe-4S] Clusters.

Thioredoxin reductases (TrxR) activate thioredoxins (Trx) that regulate the activity of diverse target proteins essential to prokaryotic and eukaryotic life. However, very little is understood of TrxR/Trx systems and redox control in methanogenic microbes from the domain Archaea (methanogens), for which genomes are abundant with annotations for ferredoxin:thioredoxin reductases [Fdx/thioredoxin reductase (FTR)] from group 4 of the widespread FTR-like family. Only two from the FTR-like family are characterized: the plant-type FTR from group 1 and FDR from group 6. Herein, the group 4 archetype (AFTR) from Methanosarcina acetivorans was characterized to advance understanding of the family and TrxR/Trx systems in methanogens. The modeled structure of AFTR, together with EPR and Mössbauer spectroscopies, supports a catalytic mechanism similar to plant-type FTR and FDR, albeit with important exceptions. EPR spectroscopy of reduced AFTR identified a transient [4Fe-4S]1+ cluster exhibiting a mixture of S = 7/2 and typical S = 1/2 signals, although rare for proteins containing [4Fe-4S] clusters, it is most likely the on-pathway intermediate in the disulfide reduction. Furthermore, an active site histidine equivalent to residues essential for the activity of plant-type FTR and FDR was found dispensable for AFTR. Finally, a unique thioredoxin system was reconstituted from AFTR, ferredoxin, and Trx2 from M. acetivorans, for which specialized target proteins were identified that are essential for growth and other diverse metabolisms.

Mechanistic understanding of acclimation and energy metabolism of acetoclastic methanogens under different substrate to microorganism ratios.

Mechanistic understanding of acetoclastic methanogenesis is pivotal for optimizing anaerobic digestion for efficient methane production. In this study, two different operational modes, continuous flow reactor (CFR) and sequencing batch reactor (SBR), accompanied with solids retention times (SRT) of 10 days (SBR10d and CFR10d) and 25 days (SBR25d and CFR25d) were implemented to elucidate their impacts on microbial communities and energy metabolism of methanogens in acetate-fed systems. Microbial community analysis revealed that the relative abundance of Methanosarcina (16.0%-46.0%) surpassed Methanothrix (3.7%-22.9%) in each reactor. SBRs had the potential to enrich both Methanothrix and Methanosarcina. Compared to SBRs, CFRs had lower total relative abundance of methanogens. Methanosarcina exhibited a superior enrichment in reactors with 10-day SRT, while Methanothrix preferred to be acclimated in reactors with 25-day SRT. The operational mode and SRT were also observed to affect the distribution of acetate-utilizing bacteria, including Pseudomonas, Desulfocurvus, Mesotoga, and Thauera. Regarding enzymes involved in energy metabolism, Ech and Vho/Vht demonstrated higher relative abundances at 10-day SRT compared to 25-day SRT, whereas Fpo and MtrA-H showed higher relative abundances in SBRs than those in CFRs. The relative abundance of genes encoding ATPase harbored by Methanothrix was higher than Methanosarcina at 25-day SRT. Additionally, the relative abundance of V/A-type ATPase (typically for methanogens) was observed higher in SBRs compared to CFRs, while the F-type ATPase (typically for bacteria) exhibited higher relative abundance in CFRs than that in SBRs.

Pyruvate-dependent growth of Methanosarcina acetivorans.

Methanogenesis is a key step during anaerobic biomass degradation. Methanogenic archaea (methanogens) are the only organisms coupling methanogenic substrate conversion to energy conservation. The range of substrates utilized by methanogens is limited, with acetate and H2+CO2 being the ecologically most relevant. The only single methanogenic energy substrate containing more carbon-carbon bonds than acetate is pyruvate. Only the aggregate-forming, freshwater methanogen Methanosarcina barkeri Fusaro was shown to grow on this compound. Here, the pyruvate-utilizing capabilities of the single-celled, marine Methanosarcina acetivorans were addressed. Robust pyruvate-dependent, methanogenic, growth could be established by omitting CO2 from the growth medium. Growth rates which were independent of the pyruvate concentration indicated that M. acetivorans actively translocates pyruvate across the cytoplasmic membrane. When 2-bromoethanesulfonate (BES) inhibited methanogenesis to more than 99%, pyruvate-dependent growth was acetogenic and sustained. However, when methanogenesis was completely inhibited M. acetivorans did not grow on pyruvate. Analysis of metabolites showed that acetogenesis is used by BES-inhibited M. acetivorans as a sink for electrons derived from pyruvate oxidation and that other, thus far unidentified, metabolites are produced.IMPORTANCEThe known range of methanogenic growth substrates is very limited and M. acetivorans is only the second methanogenic species for which growth on pyruvate is demonstrated. Besides some commonalities, analysis of M. acetivorans highlights differences in pyruvate metabolism among Methanosarcina species. The observation that M. acetivorans probably imports pyruvate actively indicates that the capabilities for heterotrophic catabolism in methanogens may be underestimated. The mostly acetogenic growth of M. acetivorans on pyruvate with concomitant inhibition of methanogenesis confirms that energy conservation of methanogenic archaea can be independent of methane formation.

Time-shifted expression of acetoclastic and methylotrophic methanogenesis by a single Methanosarcina genomospecies predominates the methanogen dynamics in Philippine rice field soil.

BACKGROUND: The final step in the anaerobic decomposition of biopolymers is methanogenesis. Rice field soils are a major anthropogenic source of methane, with straw commonly used as a fertilizer in rice farming. Here, we aimed to decipher the structural and functional responses of the methanogenic community to rice straw addition during an extended anoxic incubation (120 days) of Philippine paddy soil. The research combined process measurements, quantitative real-time PCR and RT-PCR of particular biomarkers (16S rRNA, mcrA), and meta-omics (environmental genomics and transcriptomics). RESULTS: The analysis methods collectively revealed two major bacterial and methanogenic activity phases: early (days 7 to 21) and late (days 28 to 60) community responses, separated by a significant transient decline in microbial gene and transcript abundances and CH4 production rate. The two methanogenic activity phases corresponded to the greatest rRNA and mRNA abundances of the Methanosarcinaceae but differed in the methanogenic pathways expressed. While three genetically distinct Methanosarcina populations contributed to acetoclastic methanogenesis during the early activity phase, the late activity phase was defined by methylotrophic methanogenesis performed by a single Methanosarcina genomospecies. Closely related to Methanosarcina sp. MSH10X1, mapping of environmental transcripts onto metagenome-assembled genomes (MAGs) and population-specific reference genomes revealed this genomospecies as the key player in acetoclastic and methylotrophic methanogenesis. The anaerobic food web was driven by a complex bacterial community, with Geobacteraceae and Peptococcaceae being putative candidates for a functional interplay with Methanosarcina. Members of the Methanocellaceae were the key players in hydrogenotrophic methanogenesis, while the acetoclastic activity of Methanotrichaceae members was detectable only during the very late community response. CONCLUSIONS: The predominant but time-shifted expression of acetoclastic and methylotrophic methanogenesis by a single Methanosarcina genomospecies represents a novel finding that expands our hitherto knowledge of the methanogenic pathways being highly expressed in paddy soils. Video Abstract.

Small protein mediates inhibition of ammonium transport in Methanosarcina mazei-an ancient mechanism?

IMPORTANCE: Small proteins containing fewer than 70 amino acids, which were previously disregarded due to computational prediction and biochemical detection challenges, have gained increased attention in the scientific community in recent years. However, the number of functionally characterized small proteins, especially in archaea, is still limited. Here, by using biochemical and genetic approaches, we demonstrate a crucial role of the small protein sP36 in the nitrogen metabolism of M. mazei, which modulates the ammonium transporter AmtB1 according to nitrogen availability. This modulation might represent an ancient archaeal mechanism of AmtB1 inhibition, in contrast to the well-studied uridylylation-dependent regulation in bacteria.

Unveiling the Biochar-Respiratory Growth of Methanosarcina acetivorans Involving Extracellular Polymeric Substances.

Biochar can be applied to diverse natural and engineered anaerobic systems. Biochar plays biogeochemical roles during its production, storage, and environmental dynamics, one of which is related to the global methane flux governed by methanotrophs and methanogens. Our understanding of relevant mechanisms is currently limited to the roles of biochar in methanotrophic growth, but less is known about the roles of biochar in methanogenic growth. Here, we demonstrated that biochar enhanced the methanogenic growth of a model methanogen, Methanosarcina acetivorans, and the role of biochar as an electron acceptor during methanogenic growth was confirmed, which is referred to as biochar-respiratory growth. The biochar-respiratory growth of M. acetivorans promoted the secretion of extracellular polymeric substances (EPS) with augmented electron transfer capabilities, and the removal of EPS significantly attenuated extracellular electron transfer. Identification and quantification of prosthetic cofactors for EPS suggest an important role of flavin and F420 in extracellular electron transfer. Transcriptomic analysis provided additional insights into the biochar-respiratory growth of M. acetivorans, showing that there was a positive response in transcriptional regulation to the favorable growth environment provided by biochar, which stimulated global methanogenesis. Our results shed more light on the in situ roles of biochar in the ecophysiology of methanogens in diverse anaerobic environments.

The minimal SUF system is not required for Fe-S cluster biogenesis in the methanogenic archaeon Methanosarcina acetivorans.

Iron-sulfur (Fe-S) proteins are essential for the ability of methanogens to carry out methanogenesis and biological nitrogen fixation (diazotrophy). Nonetheless, the factors involved in Fe-S cluster biogenesis in methanogens remain largely unknown. The minimal SUF Fe-S cluster biogenesis system (i.e., SufBC) is postulated to serve as the primary system in methanogens. Here, the role of SufBC in Methanosarcina acetivorans, which contains two sufCB gene clusters, was investigated. The CRISPRi-dCas9 and CRISPR-Cas9 systems were utilized to repress or delete sufC1B1 and sufC2B2, respectively. Neither the dual repression of sufC1B1 and sufC2B2 nor the deletion of both sufC1B1 and sufC2B2 affected the growth of M. acetivorans under any conditions tested, including diazotrophy. Interestingly, deletion of only sufC1B1 led to a delayed-growth phenotype under all growth conditions, suggesting that the deletion of sufC2B2 acts as a suppressor mutation in the absence of sufC1B1. In addition, the deletion of sufC1B1 and/or sufC2B2 did not affect the total Fe-S cluster content in M. acetivorans cells. Overall, these results reveal that the minimal SUF system is not required for Fe-S cluster biogenesis in M. acetivorans and challenge the universal role of SufBC in Fe-S cluster biogenesis in methanogens.

Expression of V-nitrogenase and Fe-nitrogenase in Methanosarcina acetivorans is controlled by molybdenum, fixed nitrogen, and the expression of Mo-nitrogenase.

All nitrogen-fixing bacteria and archaea (diazotrophs) use molybdenum (Mo) nitrogenase to reduce dinitrogen (N2) to ammonia, with some also containing vanadium (V) and iron-only (Fe) nitrogenases that lack Mo. Among diazotrophs, the regulation and usage of the alternative V-nitrogenase and Fe-nitrogenase in methanogens are largely unknown. Methanosarcina acetivorans contains nif, vnf, and anf gene clusters encoding putative Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase, respectively. This study investigated nitrogenase expression and growth by M. acetivorans in response to fixed nitrogen, Mo/V availability, and CRISPRi repression of the nif, vnf, and/or anf gene clusters. The availability of Mo and V significantly affected growth of M. acetivorans with N2 but not with NH4Cl. M. acetivorans exhibited the fastest growth rate and highest cell yield during growth with N2 in medium containing Mo, and the slowest growth in medium lacking Mo and V. qPCR analysis revealed the transcription of the nif operon is only moderately affected by depletion of fixed nitrogen and Mo, whereas vnf and anf transcription increased significantly when fixed nitrogen and Mo were depleted, with removal of Mo being key. Immunoblot analysis revealed Mo-nitrogenase is detected when fixed nitrogen is depleted regardless of Mo availability, while V-nitrogenase and Fe-nitrogenase are detected only in the absence of fixed nitrogen and Mo. CRISPRi repression studies revealed that V-nitrogenase and/or Fe-nitrogenase are required for Mo-independent diazotrophy, and unexpectedly that the expression of Mo-nitrogenase is also required. These results reveal that alternative nitrogenase production in M. acetivorans is tightly controlled and dependent on Mo-nitrogenase expression. IMPORTANCE Methanogens and closely related methanotrophs are the only archaea known or predicted to possess nitrogenase. Methanogens play critical roles in both the global biological nitrogen and carbon cycles. Moreover, methanogens are an ancient microbial lineage and nitrogenase likely originated in methanogens. An understanding of the usage and properties of nitrogenases in methanogens can provide new insight into the evolution of nitrogen fixation and aid in the development nitrogenase-based biotechnology. This study provides the first evidence that a methanogen can produce all three forms of nitrogenases, including simultaneously. The results reveal components of Mo-nitrogenase regulate or are needed to produce V-nitrogenase and Fe-nitrogenase in methanogens, a result not seen in bacteria. Overall, this study provides a foundation to understand the assembly, regulation, and activity of the alternative nitrogenases in methanogens.

In vivo structure probing of RNA in Archaea: novel insights into the ribosome structure of Methanosarcina acetivorans.

Structure probing combined with next-generation sequencing (NGS) has provided novel insights into RNA structure-function relationships. To date, such studies have focused largely on bacteria and eukaryotes, with little attention given to the third domain of life, archaea. Furthermore, functional RNAs have not been extensively studied in archaea, leaving open questions about RNA structure and function within this domain of life. With archaeal species being diverse and having many similarities to both bacteria and eukaryotes, the archaea domain has the potential to be an evolutionary bridge. In this study, we introduce a method for probing RNA structure in vivo in the archaea domain of life. We investigated the structure of ribosomal RNA (rRNA) from Methanosarcina acetivorans, a well-studied anaerobic archaeal species, grown with either methanol or acetate. After probing the RNA in vivo with dimethyl sulfate (DMS), Structure-seq2 libraries were generated, sequenced, and analyzed. We mapped the reactivity of DMS onto the secondary structure of the ribosome, which we determined independently with comparative analysis, and confirmed the accuracy of DMS probing in M. acetivorans Accessibility of the rRNA to DMS in the two carbon sources was found to be quite similar, although some differences were found. Overall, this study establishes the Structure-seq2 pipeline in the archaea domain of life and informs about ribosomal structure within M. acetivorans.

Genetic and Physiological Probing of Cytoplasmic Bypasses for the Energy-Converting Methyltransferase Mtr in Methanosarcina acetivorans.

Methanogenesis is a unique energy metabolism carried out by members of the domain Archaea. Unlike most other methanogens, which reduce CO2 to methane with hydrogen as the electron donor, Methanosarcina acetivorans is able to grow on methylated compounds, on acetate, and on carbon monoxide (CO). These substrates are metabolized via distinct yet overlapping pathways. For the use of any single methanogenic substrate, the membrane-integral, energy-converting N5-methyl-tetrahydrosarcinapterin (H4SPT):coenzyme M (HS-CoM) methyltransferase (Mtr) is required. It was proposed that M. acetivorans can bypass the methyl transfer catalyzed by Mtr via cytoplasmic activities. To address this issue, conversion of different energy substrates by an mtr deletion mutant was analyzed. No significant methyl transfer from H4SPT to HS-CoM could be detected with CO as the electron donor. In contrast, formation of methane and CO2 in the presence of methanol or trimethylamine was indicative of an Mtr bypass in the oxidative direction. As methane thiol and dimethyl sulfide were transiently produced during methylotrophic methanogenesis in the mtr mutant, involvement in this process of methyl sulfide-dependent methyltransferases (Mts) was analyzed in a strain lacking both the Mts system and Mtr. It could be unequivocally demonstrated that the Mts system is not involved in bypassing Mtr, thereby ruling out previous proposals. Conversion of [13C]methanol indicated that in the absence of Mtr M. acetivorans provides the reducing equivalents for methyl-S-CoM reduction to methane by oxidizing (an) intracellular compound(s) to CO2 rather than disproportioning the source of methyl groups. Thus, no in vivo Mtr bypass appears to exist in M. acetivorans. IMPORTANCE Methanogenic archaea possess only a limited number of chemiosmotic coupling sites in their respiratory chains. Among them, N5-methyl-H4SPT:HS-CoM methyltransferase (Mtr) is the most widely distributed. Previous observations led to the conclusion that Methanosarcina acetivorans is able to bypass this reaction via methyl sulfide-dependent methyltransferases (Mts). However, strains lacking Mtr are not able to produce methane from CO. Also, these strains are unable to oxidize methylated substrates to CO2, in contrast to observations in the close relative Methanosarcina barkeri. The results also highlight the sole function of the Mts system in methyl sulfide metabolism. Thus, no in vivo Mtr bypass appears to exist in M. acetivorans.

Lactate oxidation is linked to energy conservation and to oxygen detoxification via a putative terminal cytochrome oxidase in Methanosarcina acetivorans.

The marine archaeon Methanosarcina acetivorans contains a putative NAD + -independent d-lactate dehydrogenase (D-iLDH/glycolate oxidase) encoded by the MA4631 gene, belonging to the FAD-oxidase C superfamily. Nucleotide sequences similar to MA4631 gene, were identified in other methanogens and Firmicutes with >90 and 35-40% identity, respectively. Therefore, the lactate metabolism in M. acetivorans is reported here. Cells subjected to intermittent pulses of oxygen (air-adapted; AA-Ma cells) consumed lactate only in combination with acetate, increasing methane production and biomass yield. In AA-Ma cells incubated with d-lactate plus [14C]-l-lactate, the radioactive label was found in methane, CO2 and glycogen, indicating that lactate metabolism fed both methanogenesis and gluconeogenesis. Moreover, d-lactate oxidation was coupled to O2-consumption which was sensitive to HQNO; also, AA-Ma cells showed high transcript levels of gene dld and those encoding subunits A (MA1006) and B (MA1007) of a putative cytochrome bd quinol oxidase, compared to anaerobic control cells. An E. coli mutant deficient in dld complemented with the MA4631 gene, grew with d-lactate as carbon source and showed membrane-bound d-lactate:quinone oxidoreductase activity. The product of the MA4631 gene is a FAD-containing monomer showing activity of iLDH with preference to d-lactate. The results suggested that air adapted M. acetivorans is able to co-metabolize lactate and acetate with associated oxygen consumption by triggering the transcription and synthesis of the D-iLDH and a putative cytochrome bd: methanophenazine (quinol) oxidoreductase. Biomass generation and O2 consumption, suggest a potentially new oxygen detoxification mechanism coupled to energy conservation in this methanogen.

Active in vivo translocation of the Methanosarcina mazei Gö1 Casposon.

Casposons are transposable elements containing the CRISPR associated gene Cas1solo. Identified in many archaeal genomes, casposons are discussed as the origin of CRISPR-Cas systems due to their proposed Cas1solo-dependent translocation. However, apart from bioinformatic approaches and the demonstration of Cas1solo integrase and endonuclease activity in vitro, casposon transposition has not yet been shown in vivo. Here, we report on active casposon translocations in Methanosarcina mazei Gö1 using two independent experimental approaches. First, mini-casposons, consisting of a R6Kγ origin and two antibiotic resistance cassettes, flanked by target site duplications (TSDs) and terminal inverted repeats (TIRs), were generated, and shown to actively translocate from a suicide plasmid and integrate into the chromosomal MetMaz-C1 TSD IS1a. Second, casposon excision activity was confirmed in a long-term evolution experiment using a Cas1solo overexpression strain in comparison to an empty vector control under four different treatments (native, high temperature, high salt, mitomycin C) to study stress-induced translocation. Analysis of genomic DNA using a nested qPCR approach provided clear evidence of casposon activity in single cells and revealed significantly different casposon excision frequencies between treatments and strains. Our results, providing the first experimental evidence for in vivo casposon activity are summarized in a modified hypothetical translocation model.

Engineering mutually orthogonal PylRS/tRNA pairs for dual encoding of functional histidine analogues.

The availability of an expanded genetic code opens exciting new opportunities in enzyme design and engineering. In this regard histidine analogues have proven particularly versatile, serving as ligands to augment metalloenzyme function and as catalytic nucleophiles in designed enzymes. The ability to genetically encode multiple functional residues could greatly expand the range of chemistry accessible within enzyme active sites. Here, we develop mutually orthogonal translation components to selectively encode two structurally similar histidine analogues. Transplanting known mutations from a promiscuous Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRSIFGFF ) into a single domain PylRS from Methanomethylophilus alvus (MaPylRSIFGFF ) provided a variant with improved efficiency and specificity for 3-methyl-L-histidine (MeHis) incorporation. The MaPylRSIFGFF clone was further characterized using in vitro biochemical assays and x-ray crystallography. We subsequently engineered the orthogonal MmPylRS for activity and selectivity for 3-(3-pyridyl)-L-alanine (3-Pyr), which was used in combination with MaPylRSIFGFF to produce proteins containing both 3-Pyr and MeHis. Given the versatile roles played by histidine in enzyme mechanisms, we anticipate that the tools developed within this study will underpin the development of enzymes with new and enhanced functions.

Crystal Structure of Pyrrolysyl-tRNA Synthetase from a Methanogenic Archaeon ISO4-G1 and Its Structure-Based Engineering for Highly-Productive Cell-Free Genetic Code Expansion with Non-Canonical Amino Acids.

Pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). Previously, we achieved full productivity of cell-free protein synthesis for bulky non-canonical amino acids, including Nε-((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS with structure-based mutations in and around the amino acid binding pocket (first-layer and second-layer mutations, respectively). Recently, the PylRS·tRNAPyl pair from a methanogenic archaeon ISO4-G1 was used for genetic code expansion. In the present study, we determined the crystal structure of the methanogenic archaeon ISO4-G1 PylRS (ISO4-G1 PylRS) and compared it with those of structure-known PylRSs. Based on the ISO4-G1 PylRS structure, we attempted the site-specific incorporation of Nε-(p-ethynylbenzyloxycarbonyl)-L-lysine (pEtZLys) into proteins, but it was much less efficient than that of TCO*Lys with M. alvus PylRS mutants. Thus, the first-layer mutations (Y125A and M128L) of ISO4-G1 PylRS, with no additional second-layer mutations, increased the protein productivity with pEtZLys up to 57 ± 8% of that with TCO*Lys at high enzyme concentrations in the cell-free protein synthesis.

The dual role of a multi-heme cytochrome in methanogenesis: MmcA is important for energy conservation and carbon metabolism in Methanosarcina acetivorans.

Methanogenic archaea belonging to the Order Methanosarcinales conserve energy using an electron transport chain (ETC). In the genetically tractable strain Methanosarcina acetivorans, ferredoxin donates electrons to the ETC via the Rnf (Rhodobacter nitrogen fixation) complex. The Rnf complex in M. acetivorans, unlike its counterpart in Bacteria, contains a multiheme c-type cytochrome (MHC) subunit called MmcA. Early studies hypothesized MmcA is a critical component of Rnf, however recent work posits that the primary role of MmcA is facilitating extracellular electron transport. To explore the physiological role of MmcA, we characterized M. acetivorans mutants lacking either the entire Rnf complex (∆mmcA-rnf) or just the MmcA subunit (∆mmcA). Our data show that MmcA is essential for growth during acetoclastic methanogenesis but neither Rnf nor MmcA is required for methanogenic growth on methylated compounds. On methylated compounds, the absence of MmcA alone leads to a more severe growth defect compared to a Rnf deletion likely due to different strategies for ferredoxin oxidation that arise in each strain. Transcriptomic data suggest that the ∆mmcA mutant might oxidize ferredoxin by upregulating the cytosolic Wood-Ljundahl pathway for acetyl-CoA synthesis, whereas the ∆mmcA-rnf mutant may repurpose the F420 dehydrogenase complex (Fpo) to oxidize ferredoxin coupled to proton translocation. Beyond energy conservation, the deletion of rnf or mmcA leads to global transcriptional changes of genes involved in methanogenesis, carbon assimilation and regulation. Overall, our study provides systems-level insights into the non-overlapping roles of the Rnf bioenergetic complex and the associated MHC, MmcA.

Photo-regulated genetic encoding of dibenzo[c,g][1,2]diazocine on proteins via configuration switching.

We report two evolved Methanosarcina mazei pyrrolysine tRNA synthetases to genetically incorporate the isomers of dibenzo[c,g][1,2]diazocine-alanine (DBDAA) into proteins either in the dark or under regulation of 405 nm photo-stimulation. The genetic-encoded DBDAA realizes photo-tuning of enzymatic activity via the host-guest recognition of cucurbit[7]uril.

Update of the Pyrrolysyl-tRNA Synthetase/tRNAPyl Pair and Derivatives for Genetic Code Expansion.

The cotranslational incorporation of pyrrolysine (Pyl), the 22nd proteinogenic amino acid, into proteins in response to the UAG stop codon represents an outstanding example of natural genetic code expansion. Genetic encoding of Pyl is conducted by the pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA, tRNAPyl. Owing to the high tolerance of PylRS toward diverse amino acid substrates and great orthogonality in various model organisms, the PylRS/tRNAPyl-derived pairs are ideal for genetic code expansion to insert noncanonical amino acids (ncAAs) into proteins of interest. Since the discovery of cellular components involved in the biosynthesis and genetic encoding of Pyl, synthetic biologists have been enthusiastic about engineering PylRS/tRNAPyl-derived pairs to rewrite the genetic code of living cells. Recently, considerable progress has been made in understanding the molecular phylogeny, biochemical properties, and structural features of the PylRS/tRNAPyl pair, guiding its further engineering and optimization. In this review, we cover the basic and updated knowledge of the PylRS/tRNAPyl pair's unique characteristics that make it an outstanding tool for reprogramming the genetic code. In addition, we summarize the recent efforts to create efficient and (mutually) orthogonal PylRS/tRNAPyl-derived pairs for incorporation of diverse ncAAs by genome mining, rational design, and advanced directed evolution methods.