Methanosarcina Research Articles

Aligning fermentation conditions with non-canonical amino acid addition strategy is essential for Nε-((2-azidoethoxy)carbonyl)-L-lysine uptake and incorporation into the target protein

Protein engineering with non-canonical amino acids (ncAAs) holds great promises for diverse applications, however, there are still limitations in the implementation of this technology at manufacturing scale. The know-how to efficiently produce ncAA-incorporated proteins in a scalable manner is still very limited. In the present study, we incorporated the ncAA N6-[(2-azidoethoxy)carbonyl]-L-lysine (Azk) into an antigen binding fragment (Fab) in Escherichia coli. We used the orthogonal pyrrolysyl-tRNA synthetase/suppressor tRNACUAPyl pair from Methanosarcina mazei to incorporate Azk site-specifically. We characterized Azk uptake and Fab production at bench-scale under different fermentation conditions, varying timing and mode of Azk addition, Azk-to-cell ratio and induction time. Our results indicate that Azk uptake is comparatively efficient in the batch phase. We discovered that the time between Azk uptake and inducing its incorporation into the Fab must be kept short, which suggests that intracellular Azk is consumed and/or degraded. The results obtained in this study are an important step towards the development of efficient production methods for Azk-incorporated proteins in E. coli. The developed process is scalable and provides excellent yields of 2.95 mg functionalized Fab per g CDM, which corresponds to 80% of yield obtained with the wild type Fab. We also identified the cellular uptake of Azk being dependent on the physiological state of the cell as a potential bottleneck in production.

Uncovering the small proteome of Methanosarcina mazei using Ribo-seq and peptidomics under different nitrogen conditions

The mesophilic methanogenic archaeal model organism Methanosarcina mazei strain Gö1 is crucial for climate and environmental research due to its ability to produce methane. Here, we establish a Ribo-seq protocol for M. mazei strain Gö1 under two growth conditions (nitrogen sufficiency and limitation). The translation of 93 previously annotated and 314 unannotated small ORFs, coding for proteins ≤ 70 amino acids, is predicted with high confidence based on Ribo-seq data. LC-MS analysis validates the translation for 62 annotated small ORFs and 26 unannotated small ORFs. Epitope tagging followed by immunoblotting analysis confirms the translation of 13 out of 16 selected unannotated small ORFs. A comprehensive differential transcription and translation analysis reveals that 29 of 314 unannotated small ORFs are differentially regulated in response to nitrogen availability at the transcriptional and 49 at the translational level. A high number of reported small RNAs are emerging as dual-function RNAs, including sRNA154, the central regulatory small RNA of nitrogen metabolism. Several unannotated small ORFs are conserved in Methanosarcina species and overproducing several (small ORF encoded) small proteins suggests key physiological functions. Overall, the comprehensive analysis opens an avenue to elucidate the function(s) of multitudinous small proteins and dual-function RNAs in M. mazei.

Directed Evolution of Candidatus Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase for the Genetic Incorporation of Two Different Noncanonical Amino Acids in One Protein.

The genetic code expansion technique is a powerful chemical biology tool to install noncanonical amino acids (ncAAs) in proteins. As a key enzyme for this technique, pyrrolysyl-tRNA synthetase (PylRS), coupled with its cognate amber suppressor tRNAPyl, has been engineered for the genetic incorporation of more than 200 ncAAs. Using PylRS clones from different archaeal origins, two ncAAs have also been genetically encoded in one protein. In this work, we show that the C41AU mutant of tRNAPyl from Candidatus Methanomethylophilus alvus (CmatRNAPyl) is catalytically inert toward PylRS from Methanosarcina mazei (MmPylRS) but has weak activity toward PylRS from Ca. M. alvus (CmaPylRS). To improve the catalytic efficiency of CmaPylRS toward CmatRNAPyl-C41AU, we conducted a directed evolution of CMaPylRS by randomizing its coding sequence, followed by the screening of active mutant clones. After three rounds of randomization and screening, we identified 4 mutations, Y16F/N57D/E161G/N182I, that improve the catalytic efficiency of CMaPylRS toward CMatRNAPyl-C41AU. This new clone, named R3-14, coupling with CmatRNAPyl-C41AU to recognize an amber codon, has been successfully used together with an evolved MmPylRS clone, coupling with a mutant M. mazei tRNAPyl to recognize an ochre codon, to genetically incorporate two different ncAAs, N ε-(t-butoxycarbonyl)-lysine and N ε-acetyl-lysine, into one model protein.

Metagenomic insights into the enhanced methane production by hydrochar at varied propionate concentrations

Propionate is a crucial intermediate product in anaerobic digestion (AD), but its degradation is thermodynamically unfavorable. Its accumulation in the AD system can significantly reduce methane production efficiency and may lead to severe acidification. The present study investigated the methane production at various propionate concentrations (15, 30 and 60 mM) with and without hydrochar and also explored the microbial shifts by metagenomic analysis. The findings revealed that higher propionate concentrations reduced methane production and propionate degradation efficiency, leading to acetate accumulation. The addition of hydrochar mitigated the inhibitory effects of increased propionate concentrations, notably reduced the lag phase by 20.13 % and enhanced the methane production rate by 138.43 % under 30 mM propionate. This also resulted in faster degradation of propionate and acetate. Hydrochar impacted the microbial communities’ functional potentials, enhancing methane metabolic potential in all conditions. Genomic-centric analysis revealed that hydrochar selectively enriched syntrophic propionate-oxidizing bacteria (SPOB) candidate MAG79 Syntrophopropionicum sp., which could participate in DIET, across different concentrations of propionate, with the most pronounced effect at 30 mM propionate. This was distinct from the dominant species in the control groups, MAG63 Syntrophosphaera sp. and MAG69 Syntrophobacteraceae sp.. Hydrochar further supported the growth of another DIET candidate MAG4 Methanosarcina mazei and increased the abundance of proposed syntrophic acetate-oxidizing bacterium (SAOBs), across all propionate concentrations. These effects collectively led to more efficient propionate degradation and methane production.

Tuning the Functionality of Designer Translating Organelles with Orthogonal tRNA Synthetase/tRNA Pairs

Site-specific incorporation of noncanonical amino acids (ncAAs) can be realized by genetic code expansion (GCE) technology. Different orthogonal tRNA synthetase/tRNA (RS/tRNA) pairs have been developed to introduce a ncAA at the desired site, delivering a wide variety of functionalities that can be installed into selected proteins. Cytoplasmic expression of RS/tRNA pairs can cause a problem with background ncAA incorporation into host proteins. The application of orthogonally translating organelles (OTOs), inspired by the concept of phase separation, provides a solution for this issue in mammalian cells, allowing site-specific and protein-selective ncAA incorporation. So far, only Methanosarcina mazei (Mm) pyrrolysyl-tRNA synthetase (PylRS) has been used within OTOs, limiting the method’s potential. Here, we explored the implementation of four other widely used orthogonal RS/tRNA pairs with OTOs, which, to our surprise, were unsuccessful in generating mRNA-selective GCE. Next, we tested several experimental solutions and developed a new chimeric phenylalanyl-RS/tRNA pair that enables ncAA incorporation in OTOs in a site-specific and protein-selective manner. Our work reveals unaccounted design constraints in the spatial engineering of enzyme functions using designer organelles and presents a strategy to overcome those in vivo. We then discuss current limitations and future directions of in-cell engineering in general and protein engineering using GCE specifically.

Disentangling microbial niche balance and intermediates’ trade-offs for anaerobic digestion stability and regulation

Anaerobic digestion (AD) is a key technology for converting organic matters to methane-rich biogas. However, nutrient imbalance can destabilize the whole digestion. To realize stable operation of AD and improve its efficiency, this work considers a new strategy to control the intermediate concentrations of poor AD under nutrient stress. For this purpose, long-term digestion under different nutrient conditions was investigated. Results showed that the feedstock with a low C/N ratio (= 6) caused VFA accumulation (2072 ± 632 mg/L), leading to the inhibition of methane production. Employing a substrate with a higher C/N ratio (= 11) and/or adding NH4HCO3 (200 mg NH4+-N/Ladd) could alleviate the VFA inhibition, but excessive dosage of NH4HCO3 would induce ammonia inhibition. Through the established digestion balance between free ammonia nitrogen (FAN) between 0 and 25 mg/L, volatile fatty acid (VFA) 510–2100 mg/L, and alkalinity (ALK) 3300–7800 mg/L, an efficient methane yield of 150–250 mL/g VS was achieved and stable operation of AD under nutrient stress (low C/N ratio) was realized. Metabolic reconstruction between Euryarchaeota sp. MAG162, Methanosarcina mazei MAG53 and Mesotoga infera MAG119 highlighted that microbial niche balance was developed as a result of digestion balance, which is beneficial for stable operation of AD. These findings improved our understanding of the interaction mechanism between intermediates and microbial niches for stability control in AD.

A novel player of methylotrophic methanogensis pathway in boosting anaerobic digestion of corn stover with effluents recycling

Effluents recycling proves to be an effective method for enhancing the performance of anaerobic digestion system for lignocellulosic-like biomass. However, a limited understanding exists regarding the regulation mechanisms of methanogens. This study aims to systematically assess the dynamic interactions between process parameters, microbial communities, and methanogenic activity throughout the AD process of corn stover with effluents recycling. The results show a significant increase in the specific biogas yields of corn stover to 369 ± 115 mL/g VS with effluents recycling, an 87 % improvement compared to the control system. Effluents recycling can recover nitrogen and selectively enrich the abundances of microorganisms such as Herbinix hemicellulosilytica, Bacteroides paurosaccharolyticus and Methanosarcina mazei, etc. Correspondingly, the strategy improves the abundance of CAZymes, enhancing the efficient depolymerization of cellulose, glucuronoarabinoxylan, xyloglucan and pectin. Methanol and methylamines originating from enhanced pectin hydrolyzation and the biogenic amine pathway boost methylotrophic methanogenesis. This study addresses knowledge gaps in producing-pathways of precursors for methylotrophic methanogenesis and elucidates the mechanism to boost methanogenesis process of lignocellulosic-like biomass with effluents recycling.

Compiling a versatile toolbox for inducible gene expression in Methanosarcina mazei.

Methanosarcina mazei is a model organism, providing a platform to explore methanoarchaeal regulation mechanisms on the transcriptional and translational level. This study investigates and evaluates various molecular tools to allow inducible gene expression in M. mazei. (i) The TetR/TetO system was utilized to induce expression of a designed antisense RNA directed against sRNA154 allowing to increase transcripts of asRNA154 (500-fold), resulting in a significant decrease of sRNA154 levels (tetracycline-induced knockdown mutant). Strong reduction of sRNA154 was further confirmed in the knockdown mutant by up to 50-fold decreased transcript levels of the genes nifH, glnK1 , and glnA1 , the stability of which is increased by sRNA154. (ii) For translational regulation, an RNA thermometer was designed and first-ever utilized in an archaeon, inserted into the 5'-untranslated region of a reporter gene, which showed enhanced protein expression upon a temperature shift from 30°C to 40°C. (iii) The long 5'-UTR of a trimethylamine (TMA)-inducible polycistronic mRNA was evaluated and studied as a potential genetic tool for induced gene expression on the translational level. However, we discovered TMA-dependent regulation occurs most likely on the transcript level. (iv) A new selection marker (nourseothricin resistance) was established for M. mazei using the streptothricin acetyltransferase gene. Taken together, our findings provide a foundation for future exploration of genetic regulation and inducible gene expression in M. mazei and other methanoarchaea, advancing genetic studies in these organisms and enhancing their potential for biotechnology applications.

An evolved pyrrolysyl-tRNA synthetase with polysubstrate specificity expands the toolbox for engineering enzymes with incorporation of noncanonical amino acids

Aminoacyl-tRNA synthetase (aaRS) is a core component for genetic code expansion (GCE), a powerful technique that enables the incorporation of noncanonical amino acids (ncAAs) into a protein. The aaRS with polyspecificity can be exploited in incorporating additional ncAAs into a protein without the evolution of new, orthogonal aaRS/tRNA pair, which hence provides a useful tool for probing the enzyme mechanism or expanding protein function. A variant (N346A/C348A) of pyrrolysyl-tRNA synthetase from Methanosarcina mazei (MmPylRS) exhibited a wide substrate scope of accepting over 40 phenylalanine derivatives. However, for most of the substrates, the incorporation efficiency was low. Here, a MbPylRS (N311A/C313A) variant was constructed that showed higher ncAA incorporation efficiency than its homologous MmPylRS (N346A/C348A). Next, N-terminal of MbPylRS (N311A/C313A) was engineered by a greedy combination of single variants identified previously, resulting in an IPE (N311A/C313A/V31I/T56P/A100E) variant with significantly improved activity against various ncAAs. Activity of IPE was then tested toward 43 novel ncAAs, and 16 of them were identified to be accepted by the variant. The variant hence could incorporate nearly 60 ncAAs in total into proteins. With the utility of this variant, eight various ncAAs were then incorporated into a lanthanide-dependent alcohol dehydrogenase PedH. Incorporation of phenyllactic acid improved the catalytic efficiency of PedH toward methanol by 1.8-fold, indicating the role of modifying protein main chain in enzyme engineering. Incorporation of O-tert-Butyl-L-tyrosine modified the enantioselectivity of PedH by influencing the interactions between substrate and protein. Enzymatic characterization and molecular dynamics simulations revealed the mechanism of ncAAs affecting PedH catalysis. This study provides a PylRS variant with high activity and substrate promiscuity, which increases the utility of GCE in enzyme mechanism illustration and engineering.Graphical

M. mazei glutamine synthetase and glutamine synthetase-GlnK1 structures reveal enzyme regulation by oligomer modulation

Glutamine synthetases (GS) play central roles in cellular nitrogen assimilation. Although GS active-site formation requires the oligomerization of just two GS subunits, all GS form large, multi-oligomeric machines. Here we describe a structural dissection of the archaeal Methanosarcina mazei (Mm) GS and its regulation. We show that Mm GS forms unstable dodecamers. Strikingly, we show this Mm GS oligomerization property is leveraged for a unique mode of regulation whereby labile Mm GS hexamers are stabilized by binding the nitrogen regulatory protein, GlnK1. Our GS-GlnK1 structure shows that GlnK1 functions as molecular glue to affix GS hexamers together, stabilizing formation of GS active-sites. These data, therefore, reveal the structural basis for a unique form of enzyme regulation by oligomer modulation.

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

In the past decade, small open reading frames (sORFs) coding for proteins less than 70 amino acids (aa) in length have moved into the focus of science. sORFs and the corresponding small proteins have been recently identified in all three domains of life. However, the majority of small proteins remain functionally uncharacterized. While several bacterial small proteins have already been described, the number of identified and functionally characterized small proteins in archaea is still limited. In this study, we have discovered that the small protein 36 (sP36), which consists of only 61 aa, plays a critical role in regulating nitrogen metabolism in Methanosarcina mazei. The absence of sP36 significantly delays the growth of M. mazei when transitioning from nitrogen limitation to nitrogen sufficiency, as compared to the wild type. Through our in vivo experiments, we have observed that during nitrogen limitation, sP36 is dispersed throughout the cytoplasm; however, upon shifting the cells to nitrogen sufficiency, it relocates to the cytoplasmic membrane. Furthermore, an in vitro biochemical analysis clearly showed that sP36 interacts with high affinity with the ammonium transporter AmtB1 present in the cytoplasmic membrane during nitrogen limitation as well as with the PII-like protein GlnK1. Moreover, the in vivo GlnK1 interaction with AmtB1 due to nitrogen upshifts requires the presence of sP36. Based on our findings, we propose that in response to an ammonium upshift, sP36 targets the ammonium transporter AmtB1 and inhibits its activity by mediating the interaction with GlnK1. 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.

Characterization of binding constants and repair quantum yields of cyclobutane pyrimidine dimer by various class II DNA photolyase mutations

Photolyase is a flavoprotein that repairs DNA damage caused by solar ultraviolet radiation. The repair quantum yields of the class I photolyases are around 80%, and for the class II photolyases are around 50%. In this research, we designed six key residues at the active site of class II MmPL from Methanosarcina mazei to systematically characterize the binding constants and quantum yields. We used titration experiments to detect the binding affinity of MmPL wild-type and the mutants with the oligomer containing T<>T lesion. Based on the dissociation constants, we can calculate the enzyme-substrate complex concentrations and obtained the repair quantum yields. Through mutation, we unexpectedly obtained higher binding affinity and higher repair quantum yields than the wild type, suggesting the other role of some residues. These results are critical to studies of ultrafast repair dynamics to solve the repair photocycles.

Newly Established Genetic System for Functional Analysis of MetSV.

The linear chromosome of the Methanosarcina spherical virus with 10,567 bp exhibits 22 ORFs with mostly unknown functions. Annotation using common tools and databases predicted functions for a few genes like the type B DNA polymerase (MetSVORF07) or the small (MetSVORF15) and major (MetSVORF16) capsid proteins. For verification of assigned functions of additional ORFs, biochemical or genetic approaches were found to be essential. Consequently, we established a genetic system for MetSV by cloning its genome into the E. coli plasmid pCR-XL-2. Comparisons of candidate plasmids with the MetSV reference based on Nanopore sequencing revealed several mutations of yet unknown provenance with an impact on protein-coding sequences. Linear MetSV inserts were generated by BamHI restriction, purified and transformed in Methanosarcina mazei by an optimized liposome-mediated transformation protocol. Analysis of resulting MetSV virions by TEM imaging and infection experiments demonstrated no significant differences between plasmid-born viruses and native MetSV particles regarding their morphology or lytic behavior. The functionality of the genetic system was tested by the generation of a ΔMetSVORF09 mutant that was still infectious. Our genetic system of MetSV, the first functional system for a virus of methanoarchaea, now allows us to obtain deeper insights into MetSV protein functions and virus-host interactions.

Biohythane production from pineapple peel using Methanosarcina mazei enhanced with palm oil mill effluent (POME) sludge in single‐stage anaerobic digestion

AbstractBiohythane production via single‐stage anaerobic digestion (AD) is an effective way for sustainable energy recovery from lignocellulosic biomass. In this paper, biohythane was produced through the AD process from pineapple peel waste substrate using purely cultured Methanosarcina mazei with the enhancement of palm oil mill effluent (POME) sludge as the inoculum. This study focuses on the effects of the lignocellulosic pre‐treatment method, the addition of POME sludge into M. mazei culture medium as inoculum, and various operational conditions (food to microorganisms (F/M) ratios, temperature, pH) on gas production performances. The experimental results indicate that these parameters influenced the efficiency of biohythane production by producing the peak maximum biohythane production rate values (HPRmax) and (MPRmax), H2:CH4 = 1.93:0.67 L/L‐d, and biohythane yield (HY) and (MY), H2:CH4 = 1.18:0.55 mL/L‐substrate. This study demonstrates that biohythane gas (H2 + CH4 + CO2) production from pineapple waste can be accelerated by M. mazei only with the enhancement of POME sludge through single‐stage AD system under mesophilic batch process conditions.

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.

Comparison of Different Carbon Sources on Biomethane Production with Clostridium cellulovorans and Methanogens

Methane (CH4) has attracted attention as not only one of the hydrogen carriers in terms of energy density, but also synthetic natural gas. In nature, the decomposition of organic compounds is performed with bacterial ecosystems that can produce CH4. Clostridium cellulovorans as a decomposer was cultivated with pig manure (PM) as an unused biomass in this study. As a result of high-performance liquid chromatography (HPLC) analysis, while formate and lactate were decreased in the C. cellulovorans medium containing 0.5% PM, acetate and butyrate were increased in it. Accordingly, in order to compare with the effect of carbon sources for methane production, the cocultivation of C. cellulovorans and the methanogenesis of Methanosarcina mazei or microbial flora of methane production (MFMP) was carried out in the C. cellulovorans medium. As a result, only the cocultivation with C. cellulovorans and MFMP showed methane production in 0.5% acetate medium. Moreover, in comparison with a carbon source in either 1% acetate or 1% methanol medium, MFMP was only cultivated after being precultivated with 0.5% glucose medium for 12 h. The results revealed that MFMP with a 1% methanol medium produced methane approximately eight times higher than with 1% acetate medium. After cultivation with 1% acetate or 1% methanol, next-generation sequencing (NGS) analysis of MFMP was carried out. Interestingly, Methanofollis (0.211%), belonging to methanogens through the CO2 reduction pathway, was dominant in the 1% acetate medium for 72 h cultivation, while Methanosarcina siciliae (1.178%), M. barkeri (0.571%), and Methanofollis (0.490%) were major species in 1% methanol medium for 72 h cultivation. Since Methanosarcina spp. belong to acetoclasts (acetoclastic pathway), methanol could promote the growth of Methanosarcina spp., rather than acetate. Therefore, it seems that Methanosarcina spp. may play a key methanogenesis role in MFMP. Thus, these results will provide important information for low-cost biomethane production.

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.

DIET-like mutualism of Geobacter and methanogens at specific electrode potential boosts production of both methane and hydrogen from propionate

Direct interspecies electron transfer (DIET) has been demonstrated to be an efficient type of mutualism in methanogenesis. However, few studies have reported its presence in mixed microbial communities and its trigger mechanism in the natural environment and engineered systems. Here, we reported DIET-like mutualism of Geobacter and methanogens in the planktonic microbiome for the first time in anaerobic electrochemical digestion (AED) fed with propionate, potentially triggered by excessive cathodic hydrogen (56 times higher than the lowest) under the electrochemical condition. In contrast with model prediction without DIET, the highest current density and hydrogen and methane production were concurrently observed at −0.2 V where an abundance of Geobacter (49%) and extracellular electron transfer genes were identified in the planktonic microbiome via metagenomic analysis. Metagenomic assembly genomes annotated to Geobacter anodireducens were identified alongside two methanogens, Methanothrix harundinacea and Methanosarcina mazei, which were previously identified to participate in DIET. This discovery revealed that DIET-like mutualism could be triggered without external conductive materials, highlighting its potentially ubiquitous presence. Such mutualism simultaneously boosted methane and hydrogen production, thereby demonstrating the potential of AED in engineering applications.

KEGG_Extractor: An Effective Extraction Tool for KEGG Orthologs.

The KEGG Orthology (KO) database is a widely used molecular function reference database which can be used to conduct functional annotation of most microorganisms. At present, there are many KEGG tools based on the KO entries for annotating functional orthologs. However, determining how to efficiently extract and sort the annotation results of KEGG still hinders the subsequent genome analysis. There is a lack of effective measures used to quickly extract and classify the gene sequences and species information of the KEGG annotations. Here, we present a supporting tool: KEGG_Extractor for species-specific genes extraction and classification, which can output the results through an iterative keyword matching algorithm. It can not only extract and classify the amino acid sequences, but also the nucleotide sequences, and it has proved to be fast and efficient for microbial analysis. Analysis of the ancient Wood Ljungdahl (WL) pathway through the KEGG_Extractor reveals that ~226 archaeal strains contained the WL pathway-related genes. Most of them were Methanococcus maripaludis, Methanosarcina mazei and members of the Methanobacterium, Thermococcus and Methanosarcina genus. Using the KEGG_Extractor, the ARWL database was constructed, which had a high accuracy and complement. This tool helps to link genes with the KEGG pathway and promote the reconstruction of molecular networks. Availability and implementation: KEGG_Extractor is freely available from the GitHub.

Reprogramming Initiator and Nonsense Codons to Simultaneously Install Three Distinct Noncanonical Amino Acids into Proteins in E. coli.

Multiple noncanonical amino acids can be installed into proteins in E. coli using mutually orthogonal aminoacyl-tRNA synthetase and tRNA pairs. Here we describe a protocol for simultaneously installing three distinct noncanonical amino acids into proteins for site-specific bioconjugation at three sites. This method relies on an engineered, UAU-suppressing, initiator tRNA, which is aminoacylated with a noncanonical amino acid by Methanocaldococcus jannaschii tyrosyl-tRNA synthetase. Using this initiator tRNA/aminoacyl-tRNA synthetase pair, together with the pyrrolysyl-tRNA synthetase/tRNAPyl pairs from Methanosarcina mazei and Ca. Methanomethylophilus alvus, three noncanonical amino acids can be installed into proteins in response to the UAU, UAG, and UAA codons.