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PubMed1976

A biochemical basis for obligate methylotrophy: properties of a mutant of Pseudomonas AM1 lacking 2-oxoglutarate dehydrogenase.

Pseudomonas AMI is a facultative methylotroph which grows on a wide range of carbon compounds. A mutant of Pseudomonas AMI (ICT4I) grew only on C1 compounds and is thus an artificial obligate methylotroph. Measurements of activities of the components of the 2-oxoglutarate dehydrogenase complex suggest that the E2 component (dihydrolipoamide transsuccinylase) is not functional. All other tricarboxylic acid cycle enzymes were present with activities comparable to those in wild-type Pseudomonas AMI and cytochrome levels were unchanged in the mutant. Suspensions of the mutant oxidized pyruvate, lactate, beta-hydroxybutyrate, acetoacetate and 2-oxoglutarate at very low rates. By contrast, C1 compounds were oxidized at the same rate as in wild-type bacteria. Two revertants of ICT4I which regained 2-oxyoglutarate dehydrogenase activity also regained the ability to oxidize and grow on the same substrates as wild-type bacteria. It is concluded that lack of 2-oxoglutarate dehydrogenase may well be the basis of obligate methylotrophy in some bacteria.

Cell-Free System↗
PubMedDate not supplied

[Carbon assimilation pathways in the methylotrophy of Pseudomonas gazotropha].

The dynamics of label distribution was studied in the products of 14CH3OH assimilation by the cells of Pseudomonas gazotropha Z-1156. Substances to be first detected were glycolate, glycine and those of the chromatogram "start" spot. Later, the radioactivity was detected in phosphorylated compounds and glycerate. Cell extracts of Ps. gazotropha Z-1156 contained ribosephosphate isomerase, phosphoribulokinase and glyceraldehyde dehydrogenase but not ribulosediphosphate carboxylase. Distribution of the label in the products of 14CH3OH assimilation and the presence of active hydroxypyruvate reductase in the extract suggest that the serine cycle is involved in methylotrophy of Ps. gazotropha Z-1156. This suggestion is confirmed by the presence of active formate dehydrogenase, phosphoenolpyruvate carboxylase, (NADP+, Mn2+)-specific isocitrate dehydrogenase, (NAD, Mg2+)-specific malate dehydrogenase, malate lyase, and isocitrate lyase. The citric acid cycle is open at the alpha-ketoglutarate dehydrogenase system. The dry biomass of Ps. gazotropha Z-1156 contains over 70% of protein.

Carbon↗
PubMed1992

Metabolic pathways in Paracoccus denitrificans and closely related bacteria in relation to the phylogeny of prokaryotes.

Denitrification and methylotrophy in Paracoccus denitrificans are discussed. The properties of the enzymes of denitrification: the nitrate-nitrite antiporter, nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase are described. The genes for none of these proteins have yet been cloned and sequenced from P. denitrificans. A number of sequences are available for enzymes from Escherichia coli, Pseudomonas stutzeri and Pseudomonas aeruginosa. It is concluded that pathway specific c-type cytochromes are involved in denitrification. At least 40 genes are involved in denitrification. In methanol oxidation at least 20 genes are involved. In this case too pathway specific c-type cytochromes are involved. The sequence homology between the quinoproteins methanol dehydrogenase, alcoholde-hydrogenase and glucose dehydrogenase is discussed. This superfamily of proteins is believed to be derived from a common ancestor. The moxFJGI operon determines the structural components of methanol dehydrogenase and the associated c-type cytochrome. Upstream of this operon 3 regulatory proteins were found. The moxY protein shows the general features of a sensor protein and the moxX protein those of a regulatory protein. Thus a two component regulatory system is involved in both denitrification and methylotrophy. The phylogeny of prokaryotes based on 16S rRNA sequence is discussed. It is remarkable that the 16S rRNA of Thiosphaera pantotropha is identical to that of P. denitrificans. Still these bacteria show a number of differences. T. pantotropha is able to denitrify under aerobic circumstances and it shows heterotrophic nitrification. Nitrification and heterotrophic nitrification are found in species belonging to the beta-and gamma-subdivisions of purple non-sulfur bacteria. Thus the occurrence of heterotrophic nitrification in T. pantotropha, which belongs to the alpha-subdivision of purple non-sulfur bacteria is a remarkable property. Furthermore T. pantotropha contains two nitrate reductases of which the periplasmic one is supposed to be involved in aerobic denitrification. The nitrite reductase is of the Cu-type and not of the cytochrome cd1 type as in P. denitrificans. Also the cytochrome b of the Qbc complex of T. pantotropha is highly similar to its counterpart in P. denitrificans. It is hypothesized that the differences between these two organisms which both contain large megaplasmids is due to a combination of loss of genetic information and plasmid-coded properties. The distribution of a number of complex metabolic systems in eubacteria and in a number of species belonging to the alpha-group of purple non sulphur bacteria is reviewed.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteria↗
PubMed2026

Simplifying multiplex genome engineering in Saccharomyces cerevisiae with intron-mediated Random Assembly and INtegration (RAIN).

Engineering of multistep enzymatic pathways often involves extensive optimization of heterologous gene expression levels and requires cloning of promoter and open reading frames (ORFs) to generate expression cassettes. We present work on a nascent method for multiplex genome engineering in Saccharomyces cerevisiae that negates the requirement for cloning of expression cassettes. Our system, Random Assembly and INtegration (RAIN), uses intron-mediated homologous recombination (HR) for random in vivo assembly of exogenous promoter and ORF libraries, which are combined and cotransformed in a one-pot method. The libraries include consensus homology arms which target long terminal repeat regions of the Ty1 retrotransposon, providing over a hundred possible integration loci. In this way, our developmental system aims to negate the need for in vitro combinatorial cloning of promoters and ORFs to generate expression cassettes, simplifying in vitro DNA preparation before multiplex genome engineering. This paper presents findings from a series of experiments to demonstrate a proof of concept for the RAIN system. These include: the first reported use of intron-mediated assembly of promoters and ORFs for expression of a functional gene product; up to three markerless genomic integrations; and up to five integrations with antibiotic selection. We also present a number of innovations to improve integration efficiency during multiplex engineering in S. cerevisiae including: SGS1 gene knockout; disruption of heteroduplex rejection; modified Cas9 expression architecture; and overexpression of HR genes RAD52, MRE11, and RAD59. To demonstrate how our system can be used for single transformation phenotype engineering of multiple strains, we also transformed a library of methylotrophy associated genes to generate four new strains that were able to grow on a solid minimal medium with methanol as the sole additional carbon source. Our findings contribute to the ongoing efforts to improve multiplex genome engineering tools in S. cerevisiae, and provide the foundations for further development of a novel toolbox for generating useful genetic diversity for metabolic pathway engineering.

Saccharomyces cerevisiae↗
PubMed2026

Microbe Profile: Paracoccus denitrificans - a versatile model.

Paracoccus denitrificans is a metabolically versatile alphaproteobacterium first isolated in 1910 by Martinus Beijerinck. Similarities between the aerobic respiratory chain and membrane composition of P. denitrificans and those of eukaryotic mitochondria have stimulated the use of P. denitrificans as a model for oxidative phosphorylation. The organism has also been used extensively as a model for studies of denitrification, cytochrome c biogenesis, lithotrophy using thiosulphate as a source of energy, methylotrophy, and carbon metabolism more broadly. Through the application of structural biology and modern genome-based approaches, work on P. denitrificans continues to make significant contributions across multiple areas of microbiology.

Paracoccus denitrificans↗
PubMed2025

Synergic impact mechanisms of cover crop residue on Cd and As availability and native organic carbon mineralization in Cd and As co-contaminated paddy soil.

The synergic impacts of cover crop residue on heavy metal and metalloid availability and soil organic carbon (SOC) mineralization in contaminated paddy soil and the underlying microbial mechanism remain unclear. This study investigated the availability of cadmium (Cd) and arsenic (As) and mineralization of native SOC in paddy soil treated with 0, 0.4 %, 0.8 % and 1.2 % of δ13C-labeled cover crop residue (Astragalus sinicus L.) via 90-day incubation experiments, the related functional genes and functional microbial communities were analyzed using metagenomic binning assembly. Cover crop residue with addition rate from 0.4 % to 1.2 % significantly decreased available Cd by 56 %-85 % but increased available As by 39 %-66 % compared to the control treatment. Cover crop residue resulted in a positive priming effect on native SOC mineralization but benefited SOC sequestration. Cover crop residue increased the abundance of genes encoding iron reductase (mtrABC, pilA, omcB), sulfate reductase (sir, fpr), As(V) reductase (ArsC), organic carbon hydrolases, methanogenesis, and methylotrophy. Genomes associated with Chloroflexota and Bacteroidota encoded all these key pathways, and their abundance increased with cover crop residue application. Cover crop residue decreased soil Eh, dissolved crystalline iron oxides, enriched specific microorganisms, including Chloroflexota and Bacteroidota, and then synergistically promoted the decrease in Cd availability and the increase in As availability and native SOC mineralization in the examined paddy soil. These findings provided practical and feasible guidance for achieving both safe production and carbon sequestration in contaminated paddy fields, highlighting the requirement to cautious utilization of cover crop residue in As-contaminated paddy fileds.

Soil Pollutants↗
PubMed2026

Evidence of direct methane production from long-chain fatty acids by thermophilic Archaeoglobi.

Methanogenic degradation of long-chain fatty acids has traditionally been thought to occur through syntrophic partnerships between fatty acid-degrading bacteria and methanogenic archaea. However, recent genomic evidence suggests that certain archaea may independently carry out the entire process. Here we report the enrichment of an archaeon from the class Archaeoglobi, Candidatus Methanoglobus sphaerolipidus DLY3, from hot spring sediments in Tengchong, China. The results of selective enrichment, growth experiments, microscopy, stable isotope tracing, metagenomics and metatranscriptomics suggest that Ca. M. sphaerolipidus directly converts long-chain fatty acids to methane. This transformation involves the beta-oxidation pathway, the Wood-Ljungdahl pathway and methanogenic methyl-coenzyme M reductase and methyltransferase complexes-a process we term liparotrophy. In addition to oleic acid, Ca. M. sphaerolipidus is also capable of utilizing methanol as a substrate for methanogenesis. Our findings expand the known substrate range for methanogenic archaea beyond carbon dioxide reduction, acetoclastic methanogenesis, methylotrophy, methyl reduction, methoxydotrophy and the recently reported alkylotrophy.

Journal Article↗
PubMed1975

Tricarboxylic acid-cycle and related enzymes in restricted facultative methylotrophs.

The isolation is described of pure cultures of three non-methane-utilizing methylotrophic bacteria which, together with the previously described Bacillus PM6, have a very limited range of growth substrates; these organisms are designated "restricted facultative' methylotrophs. Two of these isolates, W6A and W3A1, grow only on glucose out of 50 non-C1 compounds tested, whereas the third isolate S2A1 and Bacillus PM6 grow on betaine, glucose, gluconate, alanine, glutamate, citrate and nutrient agar, but not on any of a further 56 non-C1 compounds. Crude sonic extracts of trimethylamine-grown and glucose-grown W6A and W3A1 isolates, and of trimethylamine-grown C2A1 (an obligate methylotroph) contain (i) no detectable 2-oxogltarate dehydrogenase activity, (ii) very low or zero specific activities of succinate dehydrogenase and succinyl-CoA synthetase and (iii) NAD+-dependent isocitrate dehydrogenase activity. Extracts of trimethylamine-grown PM6 and S2A1 methylotrophs have (i) very low 2-oxoglutarate dehydrogenase specific activities, (ii) comparatively high specific activities of succinate dehydrogenase, malate dehydrogenase and succinyl-CoA synthetase and (iii) NADP+-dependent isocitrate dehydrogenase activity but no NAD+-dependent isocitrate dehydrogenase activity. The activities of most of these enzymes are increased during growth on glucose, alanine, glutamate or citrate, but only very low 2-oxoglutarate dehydrogenase activities are present under all growth conditions. The restricted facultative methylotrophs grow on certain non-C1 compounds in the absence of 2-oxoglutarate dehydrogenase and, in some cases, of other enzymes of the tricarboxylic acid cycle; these lesions cannot therefore be the sole cause of obligate methylotrophy.

Bacteria↗
PubMed1990

Taxonomic studies on methylotrophic bacteria by 5S ribosomal RNA sequencing.

Nucleotide sequences of 5S ribosomal RNA (rRNA) isolated from 19 strains of Gram-negative methylotrophic bacteria were determined. Comparison of these sequences allowed construction of a tentative phylogenetic tree and showed that the bacteria analysed belong to the Proteobacteria and fell into several clusters, including obligate methanotrophs, obligate methylotrophs and several groups of facultative methylotrophs. Taxonomic relations between methylotrophic and non-methylotrophic bacteria are discussed, and the polyphyletic nature of methylotrophy as a taxonomic feature is highlighted.

Base Sequence↗
PubMed2026

Rewiring Carbon Metabolism in Bacillus methanolicus via Heterologous Phosphoketolase Expression Enhances Biomass Yield From Methanol and Reduces CO2 Loss.

Methylotrophic microbes are attractive alternatives to traditional heterotrophic production platforms, yet their efficiency is constrained by carbon loss through pyruvate decarboxylation and the oxidative branch of the RuMP cycle. The phosphoketolase (PKT) pathway provides a carbon-conserving alternative by cleaving fructose-6-phosphate and/or xylulose-5-phosphate into acetyl-phosphate, which can subsequently be converted to acetyl-coA without pyruvate decarboxylation. The remaining carbon intermediates are recycled through central metabolism to regenerate RuMP cycle intermediates without direct CO2 release. Here, we engineered this strategy in Bacillus methanolicus, a thermophilic methylotroph with strong industrial potential. We first established a versatile expression toolkit comprising inducible and constitutive promoters, benchmarked using an sfGFP reporter. Leveraging this system, we heterologously expressed the phosphoketolase B (pktB) gene from Methylotuvimicrobium buryatense 5GB1C which increased methanol-to-biomass yields by 18%-24% relative to controls and reduced biogenic CO2 production by 9%-12%. Chromosomal integration of pktB preserved these gains, demonstrating stability without reliance on plasmid-based expression. Together, these results show that PKT-driven metabolic rewiring enhances substrate yields in B. methanolicus and provides a scalable strategy to improve methylotrophic bioprocesses. This work expands the metabolic engineering toolbox for methylotrophs and highlights carbon-conserving pathway design as a key lever for advancing single carbon (C1) biomanufacturing.

Bacillus↗