Expression of divergent methyl/alkyl coenzyme M reductases from uncultured archaea.

Methanogens and anaerobic methane-oxidizing archaea (ANME) are important players in the global carbon cycle. Methyl-coenzyme M reductase (MCR) is a key enzyme in methane metabolism, catalyzing the last step in methanogenesis and the first step in anaerobic methane oxidation. Divergent mcr and mcr-like genes have recently been identified in uncultured archaeal lineages. However, the assembly and biochemistry of MCRs from uncultured archaea remain largely unknown. Here we present an approach to study MCRs from uncultured archaea by heterologous expression in a methanogen, Methanococcus maripaludis. Promoter, operon structure, and temperature were important determinants for MCR production. Both recombinant methanococcal and ANME-2 MCR assembled with the host MCR forming hybrid complexes, whereas tested ANME-1 MCR and ethyl-coenzyme M reductase only formed homogenous complexes. Together with structural modeling, this suggests that ANME-2 and methanogen MCRs are structurally similar and their reaction directions are likely regulated by thermodynamics rather than intrinsic structural differences.

Exploring the Path of Enhancing Ideological and Political Education in Universities in the Era of Big Data.

The emergence of the big data era has drastically altered people's lives and perceptions. One needs a thorough understanding of the topic to effectively apply big data's benefits to the ideological and political education work that colleges and universities carry out. By doing so, the advantages of big data can be better exploited and integrated into the educational process, enhancing the work's overall quality. To enhance the path of ideological and political education in colleges and universities, it is necessary to change according to the matter, advance according to the time, and make new changes according to the situation, and therefore, it is important to actively explore the path of ideological and political education in colleges and universities under the times. In this study, we research and analyze the opportunities and challenges facing the ideological and political education of universities in the era of big data, reexamine the subjective and objective environment in which the ideological and political education of universities is located, and explore the innovative development path of the ideological and political education of universities in the new environment. We will also encourage the innovative growth of ideological and political work in four areas, such as cultivating big data thinking innovation, working method innovation, working carrier innovation, and ideological work team construction, and conduct a ranking analysis on the significance of the exploration variables to improve the path of ideological work. The importance score measures the value of features in the construction of the ascending decision in the model, so the XGBoost algorithm is used to sort and analyze the significance of exploring variables to enhance the political and ideological work trajectory. The analysis of the experimental results shows that the innovation of working methods has greatly enhanced the conditions for carrying out ideological and political education in the new environment and has far-reaching implications and important significance for the innovation of ideological and political education in universities.

ß-Methyltryptamine Provoking the Crucial Role of Strictosidine Synthase Tyr151-OH for Its Stereoselective Pictet-Spengler Reactions to Tryptoline-type Alkaloids.

Strictosidine synthase (STR), the gate enzyme for monoterpenoid indole alkaloid biosynthesis, catalyzes the Pictet-Spengler reaction (PSR) of various tryptamine derivatives with secologanin assisted by "indole sandwich" stabilization. Continuous exploration with ß-methyltryptamine (IPA) stereoselectively delivered the C6-methylstrictosidines and C6-methylvincosides by enzymatic and nonenzymatic PSR, respectively. Unexpectedly, the first "nonindole sandwich" binding mode was witnessed by the X-ray structures of STR1-ligand complexes. Site-directed mutagenesis revealed the critical cryptic role of the hydroxyl group of Tyr151 in IPA biotransformation. Further computational calculations demonstrated the adjustable IPA position in STR1 upon the binding of secologanin, and Tyr151-OH facilitates the productive PSR binding mode via an advantageous hydrogen-bond network. Further chemo-enzymatic manipulation of C6-methylvincosides successfully resulted in the discovered antimalarial framework (IC50 = 0.92 µM).

Tuning Gene Expression by Phosphate in the Methanogenic Archaeon Methanococcus maripaludis.

Methanococcus maripaludis is a rapidly growing, hydrogenotrophic, and genetically tractable methanogen with unique capabilities to convert formate and CO2 to CH4. The existence of genome-scale metabolic models and an established, robust system for both large-scale and continuous cultivation make it amenable for industrial applications. However, the lack of molecular tools for differential gene expression has hindered its application as a microbial cell factory to produce biocatalysts and biochemicals. In this study, a library of differentially regulated promoters was designed and characterized based on the pst promoter, which responds to the inorganic phosphate concentration in the growth medium. Gene expression increases by 4- to 6-fold when the medium phosphate drops to growth-limiting concentrations. Hence, this regulated system decouples growth from heterologous gene expression without the need for adding an inducer. The minimal pst promoter is identified and contains a conserved AT-rich region, a factor B recognition element, and a TATA box for phosphate-dependent regulation. Rational changes to the factor B recognition element and start codon had no significant impact on expression; however, changes to the transcription start site and the 5' untranslated region resulted in the differential protein production with regulation remaining intact. Compared to a previous expression system based upon the histone promoter, this regulated expression system resulted in significant improvements in the expression of a key methanogenic enzyme complex, methyl-coenzyme M reductase, and the potentially toxic arginine methyltransferase MmpX.

Posttranslational Methylation of Arginine in Methyl Coenzyme M Reductase Has a Profound Impact on both Methanogenesis and Growth of Methanococcus maripaludis.

Catalyzing the key step for anaerobic production and/or oxidation of methane and likely other short-chain alkanes, methyl coenzyme M reductase (Mcr) and its homologs play a key role in the global carbon cycle. The McrA subunit possesses up to five conserved posttranslational modifications (PTMs) at its active site. It was previously suggested that methanogenesis marker protein 10 (Mmp10) could play an important role in methanogenesis. To systematically examine its physiological role, mmpX (locus tag MMP1554), the gene encoding Mmp10 in Methanococcus maripaludis, was deleted with a new genetic tool, resulting in the complete loss of the 5-C-(S)-methylarginine PTM of residue 275 in the McrA subunit. When the ΔmmpX mutant was complemented with the wild-type gene expressed by either a strong or a weak promoter, methylation was fully restored. Compared to the parental strain, maximal rates of methane formation by whole cells were reduced by 40 to 60% in the ΔmmpX mutant. The reduction in activity was fully reversed by the complement with the strong promoter. Site-directed mutagenesis of mmpX resulted in a differential loss of arginine methylation among the mutants in vivo, suggesting that activities of Mmp10 directly modulated methylation. R275 was present in a highly conserved PXRR275(A/S)R(G/A) signature sequence in McrAs. The only other protein in M. maripaludis containing a similar sequence was not methylated, suggesting that Mmp10 is specific for McrA. In conclusion, Mmp10 modulates the methyl-Arg PTM on McrA in a highly specific manner, which has a profound impact on Mcr activity.IMPORTANCE Mcr is the key enzyme in methanogenesis and a promising candidate for bioengineering the conversion of methane to liquid fuel. Our knowledge of Mcr is still limited. In terms of complexity, uniqueness, and environmental importance, Mcr is more comparable to photosynthetic reaction centers than conventional enzymes. PTMs have long been hypothesized to play key roles in modulating Mcr activity. Here, we directly link the mmpX gene to the arginine PTM of Mcr, demonstrate its association with methanogenesis activity, and offer insights into its substrate specificity and putative cofactor binding sites. This is also the first time that a PTM of McrA has been shown to have a substantial impact on both methanogenesis and growth in the absence of additional stressors.

Enantioselective Reduction of α,ß-Unsaturated Ketones and Aryl Ketones by Perakine Reductase.

This report describes the enantioselective reduction of structurally diverse α,ß-unsaturated ketones and aryl ketones by perakine reductase (PR) from Rauvolfia. This enzymatic reduction produces α-chiral allylic and aryl alcohols with excellent enantioselectivity and most of the products in satisfactory yields. Furthermore, the work demonstrates 1 mmol scale reactions for product delivery without any detrimental effect on yield and enantioselectivity. The catalytic mechanism, determined by 3D-structure-based modeling of PR and ligand complexes, is also described.

The carbon catabolite repressor CcpA mediates optimal competence development in Streptococcus oligofermentans through post-transcriptional regulation.

Natural transformation increases the genetic diversity of bacteria, but is costly and must be strictly controlled. We previously found that deletion of ccpA, a key regulator of carbon catabolite repression (CCR), reduced transformation efficiency of Streptococcus oligofermentans, the current work further investigated the regulatory mechanisms of CcpA. The competence operon comCDE is subjected to basal and autoregulatory transcription. A luciferase reporter detected a transcriptional readthrough (TRT) from the upstream tRNAArg into the comCDE operon, which was induced by L -arginine. Insertion of the Escherichia coli T1T2 terminator downstream of tRNAArg abolished TRT, and reduced the basal comCDE transcription by 77% and also the transformation efficiency. Deletion of ccpA increased tRNAArg TRT and tRNAArg -comCDE polycistronic transcript by twofold. An in vitro transcription assay determined that CcpA promoted the transcription termination of tRNAArg TRT, and RNA EMSA and SPR assays detected equal binding affinity of CcpA to both the RNA and DNA of tRNAArg . These results indicate that CcpA controls the basal comCDE transcription by post-transcriptional actions. Overexpression of comDE or its phospho-mimicking mutant comDED58E reduced transformation efficiency, indicating that excessive ComE impairs competence development. CCR-regulated competence was further confirmed by higher tRNAArg TRT but lower transformation efficiency in galactose than in glucose.

Identification and biosynthesis of 2-(1H-imidazol-5-yl) ethan-1-ol (histaminol) in methanogenic archaea.

Histaminol is a relatively rare metabolite most commonly resulting from histidine metabolism. Here we describe histaminol production and secretion into the culture broth by the methanogen Methanococcus maripaludis S2 as well as a number of other methanogens. This work is the first identification of this compound as a natural product in methanogens. Its biosynthesis from histidine was confirmed by the incorporation of 2H3-histidine into histaminol by growing cells of M. maripaludis S2. Possible functions of this molecule could be cell signaling as observed with histamine in eukaryotes or uptake of metal ions.

Methanogenesis.

Methanogenesis is an anaerobic respiration that generates methane as the final product of metabolism. In aerobic respiration, organic matter such as glucose is oxidized to CO2, and O2 is reduced to H2O. In contrast, during hydrogenotrophic methanogenesis, H2 is oxidized to H+, and CO2 is reduced to CH4. Although similar in principle to other types of respiration, methanogenesis has some distinctive features: the energy yield is very low, ≤1 ATP per methane generated, and only methanogens - organisms capable of this specialized metabolism - carry out biological methane production. Methanogens, like the process they catalyze, are similarly distinctive. Methanogens are comprised exclusively of archaea. They are obligate methane producers, that is, they do not grow using fermentation or alternative electron acceptors for respiration. Finally, methanogens are strict anaerobes and do not grow in the presence of O2. Historically, methanogenesis has been viewed as a highly specialized metabolism restricted to a narrow group of prokaryotes. However, recent developments have revealed enormous diversity within the methanogens and suggest that this metabolism is one of the most ancient on earth.

Transcriptome and Zymogram Analyses Reveal a Cellobiose-Dose Related Reciprocal Regulatory Effect on Cellulase Synthesis in Cellulosilyticum ruminicola H1.

The rumen bacterium Cellulosilyticum ruminicola H1 efficiently hydrolyzes cellulose. To gain insights into the regulatory mechanisms of cellulase synthesis, comparative transcriptome analysis was conducted for cultures grown on 2% filter paper, 0.5 and 0.05% cellobiose, and 0.5% birchwood xylan. It was found that cellulose induced a majority of (hemi)cellulases, including 33 cellulases and a cellulosomal scaffoldin (1.3- to 22.7-fold); seven endoxylanases, two mannanases, and two pectatelyases (2- to 16-fold); and pyruvate formate-lyase (PFL, 1.5- to 7-fold). Noticeably, 3- and 2.5-fold increased transcription of a cellobiohydrolase and the cellulosomal scaffoldin precursor were detected in 0.05% than in 0.5% cellobiose. Consistently, 9- and 4-fold higher specific cellobiohydrolase activities were detected in the filter paper and 0.05% cellobiose culture. SDS- and native-PAGE zymograms of cellulose-enriched proteins from the filter paper culture displayed cellulase activities, and cellulolytic "complexes" were enriched from the filter paper- and 0.05% cellobiose-cultures, but not from the 0.5% cellobiose culture. LC-MS/MS identified the cellulosomal scaffoldin precursor in the "complexes" in addition to cellulase, hemicellulase, and PFL proteins. The addition of 0.5% cellobiose, but not 0.05% cellobiose remarkably inhibited strain H1 to degrade filter paper. Therefore, this work reveals a cellobiose-dose related regulatory mechanism of cellulase synthesis by lower for induction and higher for repression, which has extended our understanding of the regulation of microbial cellulase synthesis.

[The in vitro refolding of ß-barrel outer membrane protein of gram-negative bacteria--a review].

A cell of gram-negative bacteria is surrounded by two layers of membrane, the inner membrane and the outer membrane. Proteins are the major composition of outer membrane. Many outer membrane proteins carry a trans-membrane ß-barrel structure that formed by multiple anti-parallel ß-strands connected with hydrogen bonds. These proteins can act as porins, transporters, enzymes, receptors, virulence factors and structural proteins. Therefore, their correct folding and membrane integration are important for the survival of gram-negative bacteria. Most ß-barrel outer membrane proteins could be easily expressed recombinantly and refolded in vitro under certain conditions. The in vitro folding processes could be monitored and investigated through many ways, which makes outer membrane proteins become a model system to study the effects of abiotic and biological factors on the folding of membrane proteins. In this article, the research progress on the in vitro refolding of outer membrane proteins are reviewed from the aspects of refolding methods, the factors that affect folding processes and experimental methods. Finally, the research prospects in this field are discussed.

Functionalized heterocyclic scaffolds derived from Morita-Baylis-Hillman Acetates.

Five series of heterocycles with extraordinary structural diversity have been regiospecifically synthesized from the same Morita-Baylis-Hillman Acetates (MBHAs). All four potential electrophilic sites (α, ß, γ, δ) of MBHAs are proved to be reactive.