RESUMO
Acetogenic bacteria recently attracted attention because they reduce carbon dioxide (CO2 ) with hydrogen (H2 ) to acetate or to other products such as ethanol. Besides gases, acetogens use a broad range of substrates, but conversion of the sugar alcohol mannitol has rarely been reported. We found that the thermophilic acetogenic bacterium Thermoanaerobacter kivui grew on mannitol with a specific growth rate of 0.33 h-1 to a final optical density (OD600 ) of 2.2. Acetate was the major product formed. A lag phase was observed only in cultures pre-grown on glucose, not in those pre-grown on mannitol, indicating that mannitol metabolism is regulated. Mannitol-1-phosphate dehydrogenase (MtlD) activity was observed in cell-free extracts of cells grown on mannitol only. A gene cluster (TKV_c02830-TKV_c02860) for mannitol uptake and conversion was identified in the T. kivui genome, and its involvement was confirmed by deleting the mtlD gene (TKV_c02860) encoding the key enzyme MtlD. Finally, we overexpressed mtlD, and the recombinant MtlD carried out the reduction of fructose-6-phosphate with NADH, at a high VMAX of 1235 U mg-1 at 65°C. The enzyme was thermostable for 40 min at 75°C, thereby representing the first characterized MtlD from a thermophile.
Assuntos
Manitol/metabolismo , Desidrogenase do Álcool de Açúcar/metabolismo , Thermoanaerobacter/enzimologia , Estabilidade Enzimática , Genes Bacterianos , Família Multigênica , Thermoanaerobacter/genética , Thermoanaerobacter/crescimento & desenvolvimentoRESUMO
Thermoanaerobacter kivui is one of the very few thermophilic acetogenic microorganisms. It grows optimally at 66°C on sugars but also lithotrophically with H2 + CO2 or with CO, producing acetate as the major product. While a genome-derived model of acetogenesis has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in carbon and energy metabolism have been carried out. To address this issue, we developed a method for targeted markerless gene deletions and for integration of genes into the genome of T. kivui The strain naturally took up plasmid DNA in the exponential growth phase, with a transformation frequency of up to 3.9 × 10-6 A nonreplicating plasmid and selection with 5-fluoroorotate was used to delete the gene encoding the orotate phosphoribosyltransferase (pyrE), resulting in a ΔpyrE uracil-auxotrophic strain, TKV002. Reintroduction of pyrE on a plasmid or insertion of pyrE into different loci within the genome restored growth without uracil. We subsequently studied fructose metabolism in T. kivui The gene fruK (TKV_c23150) encoding 1-phosphofructosekinase (1-PFK) was deleted, using pyrE as a selective marker via two single homologous recombination events. The resulting ΔfruK strain, TKV003, did not grow on fructose; however, growth on glucose (or on mannose) was unaffected. The combination of pyrE as a selective marker and the natural competence of the strain for DNA uptake will be the basis for future studies on CO2 reduction and energy conservation and their regulation in this thermophilic acetogenic bacterium.IMPORTANCE Acetogenic bacteria are currently the focus of research toward biotechnological applications due to their potential for de novo synthesis of carbon compounds such as acetate, butyrate, or ethanol from H2 + CO2 or from synthesis gas. Based on available genome sequences and on biochemical experiments, acetogens differ in their energy metabolism. Thus, there is an urgent need to understand the carbon and electron flows through the Wood-Ljungdahl pathway and their links to energy conservation, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Unfortunately, genetic systems have been reported for only a few acetogenic bacteria. Here, we demonstrate proof of concept for the genetic modification of the thermophilic acetogenic species Thermoanaerobacter kivui The genetic system will be used to study genes involved in biosynthesis and energy metabolism, and may further be applied to metabolically engineer T. kivui to produce fuels and chemicals.
Assuntos
Frutose/metabolismo , Genoma Bacteriano , Thermoanaerobacter/genética , Ciclo do Carbono , Metabolismo Energético/genética , Frutose/farmacologia , Deleção de Genes , Glucose/farmacologia , Recombinação Homóloga , Manose/farmacologia , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Fosfofrutoquinases/deficiência , Fosfofrutoquinases/genética , Thermoanaerobacter/efeitos dos fármacos , Thermoanaerobacter/enzimologia , Thermoanaerobacter/crescimento & desenvolvimentoRESUMO
Patients with systemic sclerosis (SSc) have a disproportionately high prevalence of reduced bone mineral density (BMD). Polymorphisms of the vitamin D receptor (VDR) gene have been associated with osteoporosis in patients with autoimmune diseases. The aim of this study was to investigate the prevalence and possible effects of VDR polymorphism on BMD and bone metabolism in patients with SSc. In patients with SSc measurement of BMD was performed using dual-energy X-ray absorptiometry. VDR polymorphisms (FokI, BsmI) were genotyped using restriction fragment length polymorphism analysis. Markers of bone metabolism (calcium, osteocalcin, ß-crosslaps) were determined. Primary endpoint was the prevalence of VDR gene polymorphisms and the association with reduced BMD. Secondary endpoints included associations between bone metabolism and VDR gene polymorphism. 79 Caucasian patients with SSc were included. Overall, 83.5% had reduced BMD (51.9% osteopenia, 31.6% osteoporosis). The prevalence of VDR gene polymorphism (73% BsmI, 77% FokI) was comparable to studies in healthy and rheumatic populations. The homozygous presence of FokI polymorphism, but not BsmI, was significantly associated with reduced axial BMD. Fokl polymorphism was significantly associated with reduced CTX levels, although changes remained within the reference limits. VDR polymorphisms can frequently be found in patients with SSc in comparable prevalence to healthy and rheumatic populations. The homozygous presence of FokI polymorphism, but not BsmI, was significantly associated with reduced axial BMD. This could be a possible contributor for the high prevalence of reduced BMD in 83.5% of patients with SSc in this study.Trial registration. DRKS00032768, date: 05.10.2023, retrospectively registered.