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Background Imbalance in cofactors causing the accumulation of intermediates in biosynthesis pathways is a frequently occurring problem in metabolic engineering when optimizing a production pathway in a microorganism. In our previous study, a single knock-out Citrobacter werkmanii ∆ dhaD was constructed for improved 1,3-propanediol (PDO) production. Instead of an enhanced PDO concentration on this strain, the gene knock-out led to the accumulation of the toxic intermediate 3-hydroxypropionaldehyde (3-HPA). The hypothesis was emerged that the accumulation of this toxic intermediate, 3-HPA, is due to a cofactor imbalance, i.e. to the limited supply of reducing equivalents (NADH). Here, this bottleneck is alleviated by rationally engineering cell metabolism to balance the cofactor supply. Results By eliminating non-essential NADH consuming enzymes (such as lactate dehydrogenase coded by ldhA , and ethanol dehydrogenase coded by adhE ) or by increasing NADH producing enzymes, the accumulation of 3-HPA is minimized. Combining the above modifications in C. werkmanii ∆ dhaD resulted in the strain C. werkmanii ∆ dhaD ∆ ldhA ∆ adhE ::ChlFRT which provided the maximum theoretical yield of 1.00 ± 0.03 mol PDO/mol glycerol when grown on glucose/glycerol (0.33 molar ratio) on flask scale under anaerobic conditions. On bioreactor scale, the yield decreased to 0.73 ± 0.01 mol PDO/mol glycerol although no 3-HPA could be measured, which indicates the existence of a sink of glycerol by a putative glycerol dehydrogenase, channeling glycerol to the central metabolism. Conclusions In this study, a multiple knock-out was created in Citrobacter species for the first time. As a result, the concentration of the toxic intermediate 3-HPA was reduced to below the detection limit and the maximal theoretical PDO yield on glycerol was reached.

In natural 1,3-propanediol (PDO) producing microorganisms such as Klebsiella pneumoniae , Citrobacter freundii and Clostridium sp., the genes coding for PDO producing enzymes are grouped in a dha cluster. This article describes the dha cluster of a novel candidate for PDO production, Citrobacter werkmanii DSM17579 and compares the cluster to the currently known PDO clusters of Enterobacteriaceae and Clostridiaceae . Moreover, we attribute a putative function to two previously unannotated ORFs, OrfW and OrfY, both in C. freundii and in C. werkmanii : both proteins might form a complex and support the glycerol dehydratase by converting cob(I)alamin to the glycerol dehydratase cofactor coenzyme B 12 . Unraveling this biosynthesis cluster revealed high homology between the deduced amino acid sequence of the open reading frames of C. werkmanii DSM17579 and those of C. freundii DSM30040 and K. pneumoniae MGH78578, i.e., 96 and 87.5 % identity, respectively. On the other hand, major differences between the clusters have also been discovered. For example, only one dihydroxyacetone kinase (DHAK) is present in the dha cluster of C. werkmanii DSM17579, while two DHAK enzymes are present in the cluster of K. pneumoniae MGH78578 and Clostridium butyricum VPI1718.

Background 1,3-propanediol (PDO) is a substantially industrial metabolite used in the polymer industry. Although several natural PDO production hosts exist, e.g. Klebsiella sp., Citrobacter sp. and Clostridium sp., the PDO yield on glycerol is insufficient for an economically viable bio-process. Enhancing this yield via strain improvement can be achieved by disconnecting the production and growth pathways. In the case of PDO formation, this approach results in a microorganism metabolizing glycerol strictly for PDO production, while catabolizing a co-substrate for growth and maintenance. We applied this strategy to improve the PDO production with Citrobacter werkmanii DSM17579 . Results Genetic tools were developed and used to create Citrobacter werkmanii DSM17579 ∆ dhaD in which dhaD, encoding for glycerol dehydrogenase, was deleted. Since this strain was unable to grow on glycerol anaerobically, both pathways were disconnected. The knock-out strain was perturbed with 13 different co-substrates for growth and maintenance. Glucose was the most promising, although a competition between NADH-consuming enzymes and 1,3-propanediol dehydrogenase emerged. Conclusion Due to the deletion of dhaD in Citrobacter werkmanii DSM17579, the PDO production and growth pathway were split. As a consequence, the PDO yield on glycerol was improved 1,5 times, strengthening the idea that Citrobacter werkmanii DSM17579 could become an industrially interesting host for PDO production.

A way to bring phosphate-saturated soils back to an environmentally safe P level is by P mining through plants. Phosphate-solubilizing bacteria (PSB) could be very useful for increasing mining efficiency over time. The goal of this research was to investigate the adaptation and performance of PSB in conditions of high total P content in soil. In the first experiment, the P-solubilizing capacity of five PSB species (three Bacillus spp. and two Pseudomonas spp.) were tested under fully controlled conditions on several growth media with different forms of insoluble phosphate (FePO 4 , AlPO 4 , or (Ca) 3 (PO 4 ) 2 ) added at different rates. The colony growth after 14 days of inoculation demonstrated that all five bacteria were able to proliferate and solubilize P on each of the tested growth media, in contradiction with the normally used technique of halo determination. In the second experiment, the same bacterial species were inoculated in pure quartz sand amended with a nutrient solution and P was added separately in an insoluble form, as Fe–P, Al–P, or Ca–P. The extractable ammonium lactate ranged from 3.2 to 6.9 and 29.0 to 40.7 mg kg −1 sand for the insoluble Al–P and Fe–P treatments, respectively. Pseudomonas putida and Bacillus brevis performed best as PSB at high P concentration where the P is fixed with Al or Fe. In the third experiment, P . putida and B . brevis were inoculated in an acidic sandy, P-saturated soil for 4 weeks. The inoculation of the PSB gave promising results in solubilizing P.

Hop-derived food supplements and beers contain the prenylflavonoids xanthohumol (X), isoxanthohumol (IX) and the very potent phyto-oestrogen (plant-derived oestrogen mimic) 8-prenylnaringenin (8-PN). The weakly oestrogenic IX can be bioactivated via O-demethylation to 8-PN. Since IX usually predominates over 8-PN, human subjects may be exposed to increased doses of 8-PN. A dietary intervention trial with fifty healthy post-menopausal Caucasian women was undertaken. After a 4 d washout period, participants delivered faeces, blank urine and breath samples. Next, they started a 5 d treatment with hop-based supplements that were administered three times per d and on the last day, a 24 h urine sample was collected. A semi-quantitative FFQ was used to estimate fat, fibre, alcohol, caffeine and theobromine intakes. The recoveries of IX, 8-PN and X in the urine were low and considerable inter-individual variations were observed. A five-fold increase in the dosage of IX without change in 8-PN concentration resulted in a significant lower IX recovery and a higher 8-PN recovery. Classification of the subjects into poor (60 %), moderate (25 %) and strong (15 %) 8-PN producers based on either urinary excretion or microbial bioactivation capacity gave comparable results. Recent antibiotic therapy seemed to affect the 8-PN production negatively. A positive trend between methane excretion and 8-PN production was observed. Strong 8-PN producers consumed less alcohol and had a higher theobromine intake. From this study we conclude that in vivoO-demethylation of IX increases the oestrogenic potency of hop-derived products.

Fertilized chicken eggs were injected with highdosesof individual polychlorinated biphenyl (PCB) congeners (0.5 μg of PCB 77, 9.8 μg of PCB 153, or 10.9 μg of PCB 180) before incubation to investigate the structure‐specific uptake of these compounds by the embryo and their accumulation in brain and liver tissue. In accordance with earlier publications, a gradual uptake and accumulation of these compounds was observed during the last week of embryonic development. The PCB uptake and distribution to the specific tissues did not appear to be structure dependent. Wet‐weight liver PCB concentrations (18, 266, and 278 ng/g at hatching for PCB 77, PCB 153, and PCB 180, respectively) were consistently two‐ to fourfold higher than carcass levels (7 ng/g of PCB 77, 117 ng/g of PCB 153, and 81 ng/g of PCB 180 at hatching). Whereas liver and carcass concentrations increased exponentially between day 13 of incubation and hatching, PCB levels in brain tissue remained unaltered (range, 0.6–1.0 ng/g of PCB 77 and 8–12 ng/g of PCB 153 and PCB 180 throughout the last week of incubation). Lipid analysis of the organs suggested that the lipid composition of brain may be an important factor explaining the low PCB accumulation in this tissue.