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Purification and properties of two fructose-6-phosphate phosphoketolases in Bifidobacterium.

Fructose-6-phosphate phosphoketolase was purified from type strains of two species of the genus Bifidobacterium: B. globosum and B. dentium. The first species has a preferred "animal" habitat, like feces of animals and rumen of cattle; the latter is harboured in "human" habitat, like feces and dental caries of man. Two electrophoretic types of phosphoketolase (F6PPK) were previously distinguished and called "animal" and "human" type according to the habitat of the bifid organism. The purified preparations of these two phosphoketolases displayed very different optimum pH range, metal activator and molecular weight; outstanding difference was found in the substrate specificity: the enzyme from B. globosum was able to split xylulose-5-P as well as fructose-6-P, whereas the phosphoketolase from B. dentium appeared to be specific for fructose-6-P.

Actinomycetales

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

Acetaldehyde: an intermediate in the formation of ethanol from glucose by lactic acid bacteria.

Group N streptococci formed acetaldehyde and ethanol from glucose. As the enzymes aldehyde dehydrogenase, phosphotransacetylase and acetate kinase were present this would enable these organisms to reduce acetyl-CoA to acetaldehyde and convert acetyl-CoA to acetyl phosphate and acetate. A pentose phosphate pathway which converted ribose-5-phosphate to glyceraldehyde-3-phosphate was also present. Acetaldehyde could not be formed via the hexose monophosphate shunt or by direct decarboxylation of pyruvate, as the enzymes phosphoketolase and alpha-carboxylase were absent. Phosphoketolase activity was induced in Streptococcus lactis subsp. diacetylactis after growth on D-xylose. Group N streptococci also contained an NAD-dependent alcohol dehydrogenase which reduced acetaldehyde to ethanol while both NAD- and NADP-dependent alcohol dehydrogenase activities were found in Leuconostoc cremoris.

Acetaldehyde

Development of linezolid and daptomycin resistance in vancomycin resistant Enterococcus faecium during antibiotic treatment.

The increasing incidence of vancomycin-resistant enterococci (VRE) over the past decade has reduced treatment options largely to linezolid and daptomycin. However, the emergence of resistance to both agents further complicates the management of VRE infections. While the mechanisms of linezolid resistance are relatively well understood, those underlying daptomycin resistance remain less clearly defined. In this study, we analyzed genomic changes associated with the development of linezolid and daptomycin resistance in initially susceptible isolates following treatment at a Danish university hospital. Phenotypic susceptibility testing and whole-genome sequencing were performed on eight isolates obtained from the same patient. We identified two distinct Enterococcus faecium clones with different mechanisms of linezolid resistance. Linezolid resistance was associated with a G2576T mutation in the 23S rRNA gene (ST80 clone) and the presence of the poxtA gene (ST3082 clone). The ST80 clone also developed daptomycin resistance during therapy. We found that daptomycin resistance might result from either a G173R substitution in a gene annotated as an "ABC transporter ATP-binding protein (LolD)" or a nonsense mutation (Q58*) in phosphoketolase, with both alterations potentially acting synergistically, but further studies are warranted to confirm if these mutations can confer resistance. Together with these findings, the study demonstrates that a single patient may harbor multiple E. faecium clones simultaneously, highlighting the risk of treatment failure if all clones are not accurately identified.

Daptomycin

Pathways of glucose metabolism in Candida 107, a lipid-accumulating yeast.

Phosphofructokinase was not detected in extracts of Candida 107 prepared in a variety of ways but was highly active in cells treated with toluene. Disruption of these cells destroyed activity of phosphofructokinase indicating that the enzyme is extremely labile. As patterns of labelling from [I-14C]glucose and [6-14C]glucose showed that 60% of glucose was metabolized via the pentose cycle, augmentation of this cycle is necessary to account for the high molar growth yields of this yeast. Phosphoketolases, reacting with xylulose 5-phosphate and fructose 6-phosphate, were found but the extent to which they contribute to glucose metabolism was not assessed.

Aldehyde-Lyases

Strict Aerobic Lifestyle and Anaerobic Survival of Bacteria: Inseparable Twins?

For many decades the existence of strict aerobic bacteria was part of every textbook. However, considering habitats like soils or surfaces, many of these microorganisms are exposed to drastic changes in oxygen tension. A simple rain shower can change oxygen diffusion rates by a factor of 10.000. Thus, for many of the so-called strict aerobic bacteria, anaerobic growth and survival strategies were discovered, mainly relying on the use of alternative electron acceptors to oxygen, redox-active metabolites, or fermentation processes generating ATP at the substrate level. Survival without growth was recognized as an important lifestyle of bacteria. With the increasing availability of genome data, many highly diverse growth and survival strategies have become apparent in bacteria. But the overall picture is far from complete. Only recently, a novel puzzle piece of the anaerobic survival strategy of the opportunistic pathogen and model bacterium Pseudomonas aeruginosa in the absence of alternative electron acceptors was elucidated. It relies on the re-wiring of carbon flux away from the Entner-Doudoroff pathway towards the pentose-phosphate pathway and use of a phosphoketolase to allow for metabolic flux while preventing nonproductive NADH formation under these fermentation conditions and for ATP generation via acetate kinase.

Anaerobiosis

Xylose, arabinose, and rhamnose fermentation by Bacteroides ruminicola.

Metabolism and growth yields of Bacteroides ruminicola grown on d-xylose, l-arabinose, and l-rhamnose were studied. Growth yields were 62, 68, and 35.5 g (dry weight) per mol of carbohydrate fermented after correction for storage polysaccharide. Experiments with [1-(14)C]arabinose indicated that pentose was fermented by a pentose phosphate cycle plus glycolysis, with some indication of a minor phosphoketolase-type pathway. The product ratios from pentose were similar to those previously described for hexose. Rhamnose was fermented mainly to 1,2-propanediol, succinate, and acetate, although the latter was quantitatively less than expected. Estimates of adenosine 5'-triphosphate (ATP) molar growth yields could not be calculated with any certainty, as ATP generation by electron transport-linked phosphorylation cannot yet be assessed. If ATP were generated by substrate-level phosphorylation reactions alone, ATP molar growth yields for xylose, arabinose, and rhamnose would be 30, 28, and 35 g/mol. If calculations are based on an assumption that two ATP are generated by electron transport-linked phosphorylation per succinate, ATP molar growth yields become 15, 14, and 22 g/mol; if the assumption is also made that the pathway of lactaldehyde reduction is coupled to production of one ATP per 1,2-propanediol by electron transport-linked phosphorylation, the ATP molar growth yield for rhamnose fermentation becomes 14 g/mol. No preference can be expressed between these alternatives at present.

Adenosine Triphosphate

Distribution of the phosphoenolpyruvate:glucose phosphotransferase system in fermentative bacteria.

A number of selected fermentative bacteria were surveyed for the presence of the phosphoenolpyruvate:glucose phosphotransferase system, with particular attention to those organisms which ferment glucose by pathways other than the Embden-Meyerhof-Parnas pathway. The phosphoenolpyruvate:glusoe phosphotransferase system was found in all homofermentative lactic acid bacteria tested that ferment glucose via the Embden-Meyerhof-Parnas pathway, but in none of a group of heterofermentative species of Lactobacillus or Leuconostoc, which ferment glucose via the phosphoketolase pathway. A phosphoenolpyruvate:glucose phosphotransferase system was also absent in Zymomonas mobilis, which ferments glucose via an anaerobic Entner-Doudoroff pathway. It thus appears that the phosphotransferase mode of glucose transport is limited to bacteria with the Embden-Meyerhof-Parnas mode of glucose fermentation.

Adenosine Triphosphate

[Dimorphism in "Ustilago cynodontis". II--Glucidic metabolism (author's transl)].

The glucidic metabolism has been studied in four strains of Ustilago cynodontis. Two of them--M1 and M7--are mycelial strains, the two others --L1 and L7--being yeast like are respectively issued from M1 and M7. The results obtained show that the choice between the different glucidic catabolism pathways takes place at the phosphofructokinase level. When the phosphofructokinase is lacking (M1) the catabolism occurs via the pentose phosphate cycle followed by the last glycolytic reactions (between triose-phosphates and pyruvic acid). When the phosphofructokinase is present it always enters into competition with a very active phosphoglucose isomerase which makes it ineffective (L1, M7, L7). In those cases the catabolism still proceeds through the pentose cycle either completely (L7) or incompletely (L1, M7), depending in the presence or the absence of transketolase. In the last case it is the phosphoketolases which carry out the conversion between fructose-5-phosphate, xylulose-6-phosphate and acetyl-phosphate.

Aldehyde-Lyases