Methanococcus Research Articles

Application of genetic code expansion to regulate the synthesis of poly(lactate-co-3-hydroxybutyrate) in Escherichia coli

Genetic codon expansion has the potential to introduce a variety of unnatural amino acids to specific sites within target proteins. In this study, genetic codon expansion was employed to regulate the enzyme expression in metabolic pathways. Firstly, a purple protein from Actinia tenebrosa was selected as the candidate to be engineered. Bringing in UAG stop codon caused premature termination of translation, while expressing orthogonal aminoacyl-tRNA synthetase and tRNA from Methanococcus jannaschii restored translation at UAG site. However, leakage expression was observed without addition of unnatural amino acids, still it can be decreased by increasing numbers of UAG mutations. Subsequently, poly(lactate-co-3-hydroxyburyrate) [P(LA-3HB)] biosynthesis pathway was constructed in Escherichia coli, and propionyl-CoA transferase was mutated to harboring one or two more stop codons. With genetic codon expansion tools, the function of propionyl-CoA transferase was restored, promoting cells to synthesize P(LA-3HB) copolymer. Moreover, the lactate monomer content was regulated ranging from 0 to 33.42 mol% by altering the addition time of inducers. Finally, the strain accumulated 27.09 g/L P(25.1 mol% LA-3HB) in 5-L bioreactor cultivation. This is the first report on metabolic engineering of polyhydroxyalkanoate biosynthesis through genetic codon expansion and would provide helpful strategies to achieve dynamic regulation of multiple metabolic pathways.

Engineering a non-oxidative glycolysis pathway in escherichia coli for high-level citramalate production

BackgroundMethyl methacrylate (MMA) is a key precursor of polymethyl methacrylate, extensively used as a transparent thermoplastic in various industries. Conventional MMA production poses health and environmental risks; hence, citramalate serves as an alternative bacterial compound precursor for MMA production. The highest citramalate titer was previously achieved by Escherichia coli BW25113. However, studies on further improving citramalate production through metabolic engineering are limited, and phage contamination is a persistent problem in E. coli fermentation.ResultsThis study aimed to construct a phage-resistant E. coli BW25113 strain capable of producing high citramalate titers from glucose. First, promoters and heterologous cimA genes were screened, and an effective biosynthetic pathway for citramalate was established by overexpressing MjcimA3.7, a mutated cimA gene from Methanococcus jannaschii, regulated by the BBa_J23100 promoter in E. coli. Subsequently, a phage-resistant E. coli strain was engineered by integrating the Ssp defense system into the genome and mutating key components of the phage infection cycle. Then, the strain was engineered to include the non-oxidative glycolysis pathway while removing the acetate synthesis pathway to enhance the supply of acetyl-CoA. Furthermore, glucose utilization by the strain improved, thereby increasing citramalate production. Ultimately, 110.2 g/L of citramalate was obtained after 80 h fed-batch fermentation. The citramalate yield from glucose and productivity were 0.4 g/g glucose and 1.4 g/(L·h), respectively.ConclusionThis is the highest reported citramalate titer and productivity in E. coli without the addition of expensive yeast extract and additional induction in fed-bath fermentation, emphasizing its potential for practical applications in producing citramalate and its derivatives.

Alkaline hydrogenotrophic methanogenesis in Methanococcus vannielii at low carbon dioxide concentrations

Capture of carbon dioxide from the atmosphere is challenging and thermodynamically expensive because of its dilute concentration (421 ppm, December 2023). Utilizing the concentrating effect of dissolving atmospheric CO2 in alkaline solution to form dissolved inorganic carbon (DIC), we explored the potential of an alkalotolerant hydrogenotrophic methanogen, Methanococcus vannielii, to capture and convert CO2 at low partial pressures to methane. Kinetic constants for CO2 reduction to CH4 were determined from experiments in serum bottles at low pCO2 at 30˚C. At pH 7, 8, and 9, the apparent KM is 0.4, 1.2, and 1.2 mM DIC and the apparent vmax is 5.6, 7.8, and 2.9 mmol CH4 L−1 OD−1 hr−1, respectively. Using atmosphere-equilibrated DIC concentrations at pH 7, 8, and 9, methane formation rates were 1.1, 2.3, and 2.1 mmol CH4 L−1 OD−1 hr−1, respectively. Our data show that alkaline hydrogenotrophic methanogenesis is an alternative to photosynthetic CO2 fixation for biological capture and conversion of CO2 at atmospheric concentrations at reasonable rates.

Methane Production from Poly(lactic Acid) (PLA) and Polyhydroxybutyrate (PHB) Biodegradable Plastics with Anaerobic Granulated Sludge

- In this study, using Methanosarcina barkeri DSM 800 and Methanococcus vannielii DSM 1224 methanogenic bacteria under mesophilic (38 ± 1oC) and thermophilic (58 ± 1oC) conditions in anaerobic granulated sludge taken from Pakmaya Yeast Factory in Izmir, Turkey; Methane production from biodegradable plastics with poly(lactic acid) (PLA) and polyhydroxybutyrate (PHB) was investigated. Effect of different operating parameters, increasing biodegradation times (from 10 days to 500 days), different inoculumsubstrate ratios (ISRs) (16, 8, 4, 2, 1) and increasing biochemical methane potential (BMP) times (between 10 day and 500 days) for the production of methane gas from PLA and PHB biodegradable plastics in anaerobic granular sludge waste; Methanosarcina barkeri DSM 800 and Methanococcus vannielii DSM 1224 methanogenic bacteria were operated during the anaerobic digestion process under anaerobic conditions at mesophilic (38 ± 1oC) and thermophilic (58 ± 1oC) experimental temperatures. PLA biodegradable plastics were operated at optimum pH=7.6. PHB biodegradable plastics were carried out at optimum pH=8.1. Predicting the biodegradation behavior of PLA and PHB biodegradable plastics with BMP tests; It is found that the ISR parameter plays a very important role. This study showed that temperature plays a key role in the aging of microorganisms (Methanosarcina barkeri DSM 800 and Methanococcus vannielii DSM 1224 methanogenic bacteria) during anaerobic digestion, the degradation of bioplastic materials (PLA and PHB) and the degradation of their natural properties. The increase in temperature from mesophilic conditions to thermophilic conditions increased the activities of methanogenic bacteria such as Methanosarcina barkeri DSM 800 and Methanococcus vannielii DSM 1224. The maximum cumulative CH4(g) production was measured at 630 NL CH4 / kgVS for PHB biodegradable plastics in anaerobic granulated sludge with inoculum culture (the mixture of Methanosarcina barkeri DSM 800 and Methanococcus vannielii DSM 1224 methanogenic bacteria), at ISR=16 value, after 100 days, at pH=8.1 and at 58±1oC, respectively. The maximum 97% biodegradation efficiency was observed for PHB biodegradable plastics after 100 days, at pH=8.1 and at 58 ± 1oC thermophilic conditions, respectively.

Methanogens acquire and bioaccumulate nickel during reductive dissolution of nickelian pyrite.

Nickel (Ni) is a key component of the active site metallocofactors of numerous enzymes required for methanogenesis, including [NiFe]-hydrogenase, carbon monoxide dehydrogenase, and methyl CoM reductase, leading to a high demand for Ni among methanogens. However, methanogens often inhabit euxinic environments that favor the sequestration of nickel as metal-sulfide minerals, such as nickelian pyrite [(Ni,Fe)S2], that have low solubilities and that are not considered bioavailable. Recently, however, several different model methanogens (Methanosarcina barkeri, Methanococcus voltae, Methanococcus maripaludis) were shown to reductively dissolve pyrite (FeS2) and to utilize dissolution products to meet iron and sulfur biosynthetic demands. Here, using M. barkeri Fusaro, and laboratory-synthesized (Ni,Fe)S2 that was physically isolated from cells using dialysis membranes, we show that trace nickel (<20 nM) abiotically solubilized from the mineral can support methanogenesis and limited growth, roughly fivefold less than the minimum concentration known to support methanogenesis. Furthermore, when provided direct contact with (Ni,Fe)S2, M. barkeri promoted the reductive dissolution of (Ni,Fe)S2 and assimilated solubilized nickel, iron, and sulfur as its sole source of these elements. Cells that reductively dissolved (Ni,Fe)S2 bioaccumulated approximately fourfold more nickel than those grown with soluble nickel and sulfide but had similar metabolic coupling efficiencies. While the mechanism for Ni uptake in archaeal methanogens is not known, homologs of the bacterial Nik uptake system were shown to be ubiquitous across methanogen genomes. Collectively, these observations indicate that (Ni,Fe)S2 is bioavailable in anoxic environments and that methanogens can convert this mineral into nickel-, iron-, and sulfur-containing metalloenzymes to support methanogenesis and growth. IMPORTANCE Nickel is an essential metal, and its availability has changed dramatically over Earth history due to shifts in the predominant type of volcanism in the late Archean that limited its availability and an increase in euxinic conditions in the early Proterozoic that favored its precipitation as nickel sulfide minerals. Observations presented herein indicate that the methanogen, Methanosarcina barkeri, can acquire nickel at low concentration (<20 nM) from soluble and mineral sources. Furthermore, M. barkeri was shown to actively reduce nickelian pyrite; use dissolution products to meet their iron, sulfur, and nickel demands; and bioaccumulate nickel. These data help to explain how M. barkeri (and possibly other methanogens and anaerobes) can acquire nickel in contemporary and past anoxic or euxinic environments.

Colorectal Cancer Archaeome: A Metagenomic Exploration, Tunisia.

Colorectal cancer (CRC) is a serious public health problem known to have a multifactorial etiology. The association between gut microbiota and CRC has been widely studied; however, the link between archaea and CRC has not been sufficiently studied. To investigate the involvement of archaea in colorectal carcinogenesis, we performed a metagenomic analysis of 68 formalin-embedded paraffin fixed tissues from tumoral (n = 33) and healthy mucosa (n = 35) collected from 35 CRC Tunisian patients. We used two DNA extraction methods: Generead DNA FFPE kit (Qiagen, Germantown, MD, USA) and Chelex. We then sequenced the samples using Illumina Miseq. Interestingly, DNA extraction exclusively using Chelex generated enough DNA for sequencing of all samples. After data filtering and processing, we reported the presence of archaeal sequences, which represented 0.33% of all the reads generated. In terms of abundance, we highlighted a depletion in methanogens and an enrichment in Halobacteria in the tumor tissues, while the correlation analysis revealed a significant association between the Halobacteria and the tumor mucosa (p < 0.05). We reported a strong correlation between Natrialba magadii, Sulfolobus acidocaldarius, and tumor tissues, and a weak correlation between Methanococcus voltae and healthy adjacent mucosa. Here, we demonstrated the feasibility of archaeome analysis from formol fixed paraffin-embedded (FFPE) tissues using simple protocols ranging from sampling to data analysis, and reported a significant association between Halobacteria and tumor tissues in Tunisian patients with CRC. The importance of our study is that it represents the first metagenomic analysis of Tunisian CRC patients' gut microbiome, which consists of sequencing DNA extracted from paired tumor-adjacent FFPE tissues collected from CRC patients. The detection of archaeal sequences in our samples confirms the feasibility of carrying out an archaeome analysis from FFPE tissues using a simple DNA extraction protocol. Our analysis revealed the enrichment of Halobacteria, especially Natrialba magadii, in tumor mucosa compared to the normal mucosa in CRC Tunisian patients. Other species were also associated with CRC, including Sulfolobus acidocaldarius and Methanococcus voltae, which is a methanogenic archaea; both species were found to be correlated with adjacent healthy tissues.

Dihydroorotase from Methanococcus jannaschii with substrate and product bound

Dihydroorotase from <i>Methanococcus jannaschii</i> with substrate and product bound

Minimizing the Anticodon-Recognized Loop of Methanococcus jannaschii Tyrosyl-tRNA Synthetase to Improve the Efficiency of Incorporating Noncanonical Amino Acids.

In the field of genetic code expansion (GCE), improvements in the efficiency of noncanonical amino acid (ncAA) incorporation have received continuous attention. By analyzing the reported gene sequences of giant virus species, we noticed some sequence differences at the tRNA binding interface. On the basis of the structural and activity differences between Methanococcus jannaschii Tyrosyl-tRNA Synthetase (MjTyrRS) and mimivirus Tyrosyl-tRNA Synthetase (MVTyrRS), we found that the size of the anticodon-recognized loop of MjTyrRS influences its suppression activity regarding triplet and specific quadruplet codons. Therefore, three MjTyrRS mutants with loop minimization were designed. The suppression of wild-type MjTyrRS loop-minimized mutants increased by 1.8-4.3-fold, and the MjTyrRS variants enhanced the activity of the incorporation of ncAAs by 15-150% through loop minimization. In addition, for specific quadruplet codons, the loop minimization of MjTyrRS also improves the suppression efficiency. These results suggest that loop minimization of MjTyrRS may provide a general strategy for the efficient synthesis of ncAAs-containing proteins.

"Not-so-popular" orthogonal pairs in genetic code expansion.

During the past decade, genetic code expansion has been proved to be a powerful tool for protein studies and engineering. As the key part, a series of orthogonal pairs have been developed to site-specifically incorporate hundreds of noncanonical amino acids (ncAAs) into proteins by using bacteria, yeast, mammalian cells, animals, or plants as hosts. Among them, the pair of tyrosyl-tRNA synthetase/tRNATyr from Methanococcus jannaschii and the pair of pyrrolysyl-tRNA synthetase/tRNAPyl from Methanosarcina species are the most popular ones. Recently, other "not-so-popular" orthogonal pairs have started to attract attentions, because they can provide more choices of ncAA candidates and are necessary for simultaneous incorporation of multiple ncAAs into a single protein. Here, we summarize the development and applications of those "not-so-popular" orthogonal pairs, providing guidance for studying and engineering proteins.

Abstract 2628: Characterization of a FeoC-like protein in Methanococcus voltae

Abstract 2628: Characterization of a FeoC-like protein in Methanococcus voltae

Archaella Isolation.

Swimming archaea are propelled by a filamentous structure called the archaellum. The first step for the structural characterization of this filament is its isolation. Here we provide various methods that allow for the isolation of archaella filaments from well-studied archaeal model organisms. Archaella filaments have been successfully extracted from organisms belonging to different archaeal phyla, e.g., euryarchaeal methanogens such as Methanococcus voltae, and crenarchaeal hyperthermoacidophiles like Sulfolobus acidocaldarius. The filament isolation protocols that we provide in this chapter follow one of two strategies: either the filaments are sheared or extracted from whole cells by detergent extraction, prior to further final purification by centrifugation methods.

Enzyme Kinetics of Recombinant Dihydroorotase from Methanococcus jannaschii

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamoyl-L aspartate (CA) to L-dihydroorotate (DHO) in the third step of de novo pyrimidine biosynthesis. The reaction is pH dependent; at low pH the biosynthetic reaction is favored (CA to DHO) and at high pH the degradative reaction is favored (DHO to CA). Even though DHOases share a common catalytic mechanism, they form a very diverse family of proteins. Methanococcus jannaschii is a hyperthermophilic and barophilic archaeon and its DHOase (Mj DHOase) is the first archaeal one that is being studied. A previously conducted physicochemical characterization of Mj DHOase gave information into its similarities and differences from the other known DHOases. In that study, enzyme kinetics were only analyzed in the degradative direction. The purpose of the current work is to further characterize Mj DHOase by studying the kinetics in the biosynthetic direction and the dependence of the reaction on pH in both directions. The properties obtained are compared with other known DHOases. The specific activity of Mj DHOase in the biosynthetic direction is approximately half the specific activity in the degradative direction, similar to human DHOase. Mj DHOase exhibits the characteristic pH dependence of the reaction.

Towards Engineering an Orthogonal Protein Translation Initiation System.

In the last two decades, methods to incorporate non-canonical amino acids (ncAAs) into specific positions of a protein have advanced significantly; these methods have become general tools for engineering proteins. However, almost all these methods depend on the translation elongation process, and strategies leveraging the initiation process have rarely been reported. The incorporation of a ncAA specifically at the translation initiation site enables the installation of reactive groups for modification at the N-termini of proteins, which are attractive positions for introducing abiological groups with minimal structural perturbations. In this study, we attempted to engineer an orthogonal protein translation initiation system. Introduction of the identity elements of Escherichia coli initiator tRNA converted an engineered Methanococcus jannaschii tRNATyr into an initiator tRNA. The engineered tRNA enabled the site-specific incorporation of O-propargyl-l-tyrosine (OpgY) into the amber (TAG) codon at the translation initiation position but was inactive toward the elongational TAG codon. Misincorporation of Gln was detected, and the engineered system was demonstrated only with OpgY. We expect further engineering of the initiator tRNA for improved activity and specificity to generate an orthogonal translation initiation system.

Novel methanotrophic and methanogenic bacterial communities from diverse ecosystems and their impact on environment

Methane oxidizing and methane releasing bacterial communities are very pioneer microbial population, abundantly present in several ecosystems. The microbial dynamics and metabolic assemblages are positively affected by these peculiar groups of bacteria, playing very important role in sustaining carbon geochemical cycle. A number of conventional methanotrophic and methanogenic communities are reported from marine and coastal environment, forest/desert ecosystem, paddy field soil, fresh water lakes, swamps and other soil ecosystems. In rice fields, both bacterial populations are complementary to each other. Methane gas is released by one community while utilized by another community through oxidation process. The present review describe novel species belonging to methanotrophic and methanogenic bacterial communities obtained through culture dependent and culture independent approach mingled with polyphasic taxonomic methods. The novel species of methanogens and methanotrophic bacterial communities have been isolated from different ecosystems and characterized for diverse biotechnological applications. To name a few, Methylobacterium phyllosphaerae sp. nov., Methanococcus aeolicus sp. nov. and Methylocella silvestris sp. nov. have been isolated from paddy field, marine environment and forest ecosystem respectively. The present review deals with biodiversity of novel species of methanogens and methanotrophs and their impact on environment.

Identification of a novel N-linked glycan on the archaellins and S-layer protein of the thermophilic methanogen, Methanothermococcus thermolithotrophicus

Motility in archaea is facilitated by a unique structure termed the archaellum. N-Glycosylation of the major structural proteins (archaellins) is important for their subsequent incorporation into the archaellum filament. The identity of some of these N-glycans has been determined, but archaea exhibit extensive variation in their glycans, meaning that further investigations can shed light not only on the specific details of archaellin structure and function, but also on archaeal glycobiology in general. Here we describe the structural characterization of the N-linked glycan modifications on the archaellins and S-layer protein of Methanothermococcus thermolithotrophicus, a methanogen that grows optimally at 65 °C. SDS-PAGE and MS analysis revealed that the sheared archaella are composed principally of two of the four predicted archaellins, FlaB1 and FlaB3, which are modified with a branched, heptameric glycan at all N-linked sequons except for the site closest to the N termini of both proteins. NMR analysis of the purified glycan determined the structure to be α-d-glycero-d-manno-Hep3OMe6OMe-(1-3)-[α-GalNAcA3OMe-(1-2)-]-β-Man-(1-4)-[β-GalA3OMe4OAc6CMe-(1-4)-α-GalA-(1-2)-]-α-GalAN-(1-3)-β-GalNAc-Asn. A detailed investigation by hydrophilic interaction liquid ion chromatography-MS discovered the presence of several, less abundant glycan variants, related to but distinct from the main heptameric glycan. In addition, we confirmed that the S-layer protein is modified with the same heptameric glycan, suggesting a common N-glycosylation pathway. The M. thermolithotrophicus archaellin N-linked glycan is larger and more complex than those previously identified on the archaellins of related mesophilic methanogens, Methanococcus voltae and Methanococcus maripaludis This could indicate that the nature of the glycan modification may have a role to play in maintaining stability at elevated temperatures.

Tumor microenvironment targeting with dual stimuli-responsive nanoparticles based on small heat shock proteins for antitumor drug delivery

Tumour microenvironment (TME)-targeting nanoparticles (NPs) were developed based on Methanococcus jannaschii small heat shock proteins (Mj-sHSPs). Transactivator of transcription (TAT) were modified on the surface of Mj-sHSPs (T-HSPs) to enhance their cellular internalization ability (CIA), and a pH/enzyme dual sensitive PEG/N-(2-aminoethyl)piperidine-hyaluronic acid (PAHA) coat was combined with T-HSPs (PT-HSPs). PT-HSP NPs exhibited multi-layered morphologies and good stability against plasma protein adsorption. The release of paclitaxel (PTX) from PT-HSP NPs was negligible at physiological pH. Under conditions similar to the TME (acidic pH and overexpressed hyaluronidase (HAase)), the PAHA coat deshielded from PT-HSP NPs because of two factors: charge reversal and HAase degradation. Once the PAHA coat was shed, the size of the NPs decreased; its surface charge became positive; and remarkable drug release was triggered. Cellular experiments indicated that the CIA of PT-HSPs was shielded in the microenvironment of normal cells and recovered in that of tumour cells. In vivo imaging exhibited that the PT-HSP NPs had an impressive tumour targeting ability compared with the uncoated controls. The antitumor efficacy in vivo demonstrated that tumour-bearing mice treated with PTX-loaded PT-HSP NPs achieved better anti-tumour effects and safety than the Taxol formulation. In summary, this study provided Mj-sHSP NPs with coats that could be shed in response to the particular pH and enzymes in the TME, which improved the efficacy of tumour therapy. Statement of significanceThis study reports on tumor microenvironment-targeting protein-based nanoparticles (PT-HSP NPs) for targeted tumor therapy. The NPs had a multilayered structure: a protein cage, a TAT cationic layer, and a dual-sensitive coat. PT-HSP NPs exhibited multilayered morphology, with good stability against plasma protein adsorption, and PTX release negligible at physiological pH. Under the tumor microenvironment (acidic pH and overexpressed HAase), PAHA coat deshielded from PT-HSP NPs due to two factors: the charge reversal induced by protonation of piperidines in PAHA and HAase degradation. The results of cellular uptake, cytotoxicity, in vivo imaging, and tumor inhibition experiments confirmed that PT-HSP NPs exhibited promising tumor targeting efficacy in vitro and in vivo.

Structural and related studies on Mevo lectin from Methanococcus voltae A3: the first thorough characterization of an archeal lectin and its interactions.

Crystallographic and solution studies of Mevo lectin and its complexes, the first effort of its kind on an archeal lectin, reveal a structure similar to β-prism I fold lectins from plant and animal sources, but with a quaternary association involving a ring structure with seven-fold symmetry. Each subunit in the heptamer carries one sugar binding site on the first Greek key motif. The oligomeric interface is primarily made up of a parallel β-sheet involving a strand of Greek key I of one subunit and Greek key ΙΙΙ from a neighboring subunit. The crystal structures of the complexes of the lectin with mannose, αMan(1,2)αMan, αMan(1,3)αMan, a mannotriose and a mannopentose revealed a primary binding site similar to that found in other mannose specific β-prism I fold lectins. The complex with αMan(1,3)αMan provides an interesting case in which a few subunits have the reducing end at the primary binding site, while the majority have the nonreducing end at the primary binding site. The structures of complexes involving the trisaccharide and the pentasaccharide exhibit cross-linking among heptameric molecules. The observed arrangements may be relevant to the multivalency of the lectin. Phylogenetic analysis of amino acid sequences indicates that Mevo lectin is closer to β-prism I fold animal lectins than with those of plant origin. The results presented here reinforce the conclusion regarding the existence of lectins in all three domains of life. It would also appear that lectins evolved to the present form before the three domains diverged.

Structure of the ribosomal P stalk base in archaean Methanococcus jannaschii

Complexes of archaeal ribosomal proteins uL11 and uL10/P0 (the two-domain N-terminal fragment of uL10, uL10NTF/P0NTF) with the adjacent 74 nucleotides of 23S rRNA fragment (23SrRNA(74)) from Methanococcus jannaschii (Mja) were obtained, crystallized and their structures were studied. The comparative structural analysis of the complexes of Mja uL10NTF•23SrRNA(74) and Mja uL10NTF•uL11•23SrRNA(74) shows that the insertion of uL11 in the binary complex does not change the conformation of the 23S rRNA fragment. On the other hand, the interaction with this specific RNA fragment leads to the restructuring of uL11 compared to the structure of this protein in the free state. Besides, although analysis confirmed the mobility of uL10/P0 domain II, disproved the assumption that it may be in contact with rRNA or uL11. In addition, the Mja uL10NTF•uL11•23SrRNA(74) complex was cocrystallized with the antibiotic thiostrepton, and the structure of this complex was solved. The thiostrepton binding site in this archaeal complex was found between the 23S rRNA and the N-terminal domain (NTD) of the Mja uL11 protein, similar to its binding site in the one of bacterial ribosome complex with thiostrepton. Upon binding of thiostrepton, the NTD of uL11 shifts toward rRNA by 7 Å. Such a shift may be the cause of the inhibitory effect of the antibiotic on the recruitment of translation factors to the GTPase-activating region in archaeal ribosomes, similar to its inhibitory effect on protein synthesis in bacterial ribosomes.

Crystal structures and RNA-binding properties of Lsm proteins from archaea Sulfolobus acidocaldarius and Methanococcus vannielii: Similarity and difference of the U-binding mode

Sm and Sm-like (Lsm) proteins are considered as an evolutionary conserved family involved in RNA metabolism in organisms from bacteria and archaea to human. Currently, the function of Sm-like archaeal proteins (SmAP) is not well understood. Here, we report the crystal structures of SmAP proteins from Sulfolobus acidocaldarius and Methanococcus vannielii and a comparative analysis of their RNA-binding sites. Our data show that these SmAPs have only a uridine-specific RNA-binding site, unlike their bacterial homolog Hfq, which has three different RNA-binding sites. Moreover, variations in the amino acid composition of the U-binding sites of the two SmAPs lead to a difference in protein affinity for oligo(U) RNA. Surface plasmon resonance data and nucleotide-binding analysis confirm the high affinity of SmAPs for uridine nucleotides and oligo(U) RNA and the reduced affinity for adenines, guanines, cytidines and corresponding oligo-RNAs. In addition, we demonstrate that MvaSmAP1 and SacSmAP2 are capable of melting an RNA hairpin and, apparently, promote its interaction with complementary RNA.

Enhanced production of Dopa-incorporated mussel adhesive protein using engineered translational machineries.

Mussel adhesive proteins (MAPs) have great potential as bioglues, particularly in wet conditions. Although in vivo residue-specific incorporation of 3,4-dihydroxyphenylalanine (Dopa) in tyrosine-auxotrophic Escherichia coli cells allows for production of Dopa-incorporated bioengineered MAPs (dMAPs), the low production yield hinders the practical application of dMAPs. This low production yield of dMAPs is due to low translational activity of a noncanonical amino acid, Dopa, in E. coli cells. Herein, to enhance the production yield of dMAPs, we investigated the coexpression of Dopa-recognizing tyrosyl-tRNA synthetases (TyrRSs). To use the Dopa-specific Methanococcus jannaschii TyrRS (MjTyrRS-Dopa), we altered the anticodon of tyrosyl-tRNA amber suppressor into AUA (MjtRNATyrAUA ) to recognize a tyrosine codon (AUA). Co-overexpression of MjTyrRS-Dopa and MjtRNATyrAUA increased the production yield of Dopa-incorporated MAP foot protein type 3 (dfp-3) by 57%. Similarly, overexpression of E. coli TyrRS (EcTyrRS) led to a 72% higher production yield of dfp-3. Even with coexpression of Dopa-recognizing TyrRSs, dfp-3 has a high Dopa incorporation yield (over 90%) compared to ones prepared without TyrRS coexpression.