Publications

Abstract (Expand)

We described an efficient in situ generation of hydroxypyruvate from d‐serine catalyzed by a d‐amino acid oxidase from Rhodotorula gracilis. This strategy revealed an interesting alternative to the conventional chemical synthesis of hydroxypyruvate starting from toxic bromopyruvate or to the enzymatic transamination from l‐serine requiring an additional substrate as amino acceptor. Hydroxypyruvate thus produced was used as donor substrate of transketolases from Escherichia coli or from Geobacillus stearothermophilus catalyzing the stereoselective formation of a carbon−carbon bond. The enzymatic cascade reaction was performed in one‐pot in the presence of d‐serine and appropriate aldehydes for the synthesis of valuable (3S)‐hydroxyketones, which were obtained with high enantio‐ and diastereoselectivity and in good yield. The efficiency of the process was based on the irreversibility of both reactions allowing complete conversion of d‐serine and aldehydes.

Authors: None

Date Published: 6th Jun 2019

Journal: Volume361, Issue11 Special Issue: Biocatalysis, Pages 2550-2558

Abstract (Expand)

The thermoacidophilic Crenarchaeon Sulfolobus solfataricus is a model organism for archaeal adaptation to extreme environments and renowned for its ability to degrade a broad variety of substrates. It has been well characterised concerning the utilisation of numerous carbohydrates as carbon source. However, its amino acid metabolism, especially the degradation of single amino acids, is not as well understood. In this work, we performed metabolic modelling as well as metabolome, transcriptome and proteome analysis on cells grown on caseinhydrolysate as carbon source in order to draw a comprehensive picture of amino acid metabolism in S. solfataricus P2. We found that 10 out of 16 detectable amino acids are imported from the growth medium. Overall, uptake of glutamate, methionine, leucine, phenylalanine and isoleucine was the highest of all observed amino acids. Our simulations predict an incomplete degradation of leucine and tyrosine to organic acids, and in accordance with this, we detected the export of branched-chain and aromatic organic acids as well as amino acids, ammonium and trehalose into the culture supernatants. The branched-chain amino acids as well as phenylalanine and tyrosine are degraded to organic acids via oxidative Stickland reactions. Such reactions are known for prokaryotes capable of anaerobic growth, but so far have never been observed in an obligate aerobe. Also, 3-methyl-2-butenoate and 2-methyl-2-butenoate are for the first time found as products of modified Stickland reactions for the degradation of branched-chain amino acids. This work presents the first detailed description of branched-chain and aromatic amino acid catabolism in S. solfataricus.

Authors: Helge Stark, Jacqueline Wolf, Andreas Albersmeier, Trong Khoa Pham, Julia D. Hofmann, Bettina Siebers, Jörn Kalinowski, Phil Wright, Meina Neumann-Schaal, Dietmar Schomburg

Date Published: 29th May 2017

Journal: FEBS J

Abstract

Not specified

Authors: None

Date Published: 25th Jul 2013

Journal: PLoS Comput Biol

Abstract

Not specified

Authors: Adrienne Zaprasis, J. Brill, M. Thuring, G. Wunsche, M. Heun, H. Barzantny, Tamara Hoffmann, Erhard Bremer

Date Published: 28th Dec 2012

Journal: Applied and Environmental Microbiology

Abstract (Expand)

Glycine betaine is an effective osmoprotectant for Bacillus subtilis. Its import into osmotically stressed cells led to the build-up of large pools, whose size was sensitively determined by the degree of the imposed osmotic stress. The amassing of glycine betaine caused a repression in the formation of an osmostress-adaptive pool of proline, the only osmoprotectant that B. subtilis can synthesize de novo. The ABC transporter OpuA is the main glycine betaine uptake system of B. subtilis. Expression of opuA was up-regulated in response to both sudden and sustained increases in the external osmolarity. Non-ionic osmolytes exerted a stronger inducing effect on transcription than ionic osmolytes, and this was reflected in the development of corresponding OpuA-mediated glycine betaine pools. Primer extension analysis and site-directed mutagenesis pinpointed the osmotically controlled opuA promoter. Deviations from the consensus sequence of SigA-type promoters serve to keep the transcriptional activity of the opuA promoter low in the absence of osmotic stress. Expression of opuA was down regulated in a finely tuned manner in response to increases in the intracellular glycine betaine pool, regardless whether this osmoprotectant was imported or newly synthesized from choline. Such an effect was also exerted by carnitine, an effective osmoprotectant for B. subtilis that is not a substrate for the OpuA transporter. opuA expression was up-regulated in a B. subtilis mutant unable to synthesize proline in response to osmotic stress. Collectively, our data suggest that the intracellular solute pool is a key determinant for the osmotic control of opuA expression.

Authors: Tamara Hoffmann, Annette Wensing, Margot Brosius, Leif Steil, Uwe Voelker, Erhard Bremer

Date Published: 24th Nov 2012

Journal: J. Bacteriol.

Abstract (Expand)

The respiratory chain of Escherichia coli contains three quinones. Menaquinone and demethylmenaquinone have low midpoint potentials and are involved in anaerobic respiration, while ubiquinone, which has a high midpoint potential, is involved in aerobic and nitrate respiration. Here, we report that demethylmenaquinone plays a role not only in trimethylaminooxide-, dimethylsulfoxide- and fumarate-dependent respiration, but also in aerobic respiration. Furthermore, we demonstrate that demethylmenaquinone serves as an electron acceptor for oxidation of succinate to fumarate, and that all three quinol oxidases of E. coli accept electrons from this naphtoquinone derivative.

Authors: Poonam Sharma, Joost Teixeira De Mattos, Klaas J. Hellingwerf, Martijn Bekker

Date Published: 1st Sep 2012

Journal: Not specified

Abstract (Expand)

BACKGROUND: Systems biology approaches to study metabolic switching in Streptomyces coelicolor A3(2) depend on cultivation conditions ensuring high reproducibility and distinct phases of culture growth and secondary metabolite production. In addition, biomass concentrations must be sufficiently high to allow for extensive time-series sampling before occurrence of a given nutrient depletion for transition triggering. The present study describes for the first time the development of a dedicated optimized submerged batch fermentation strategy as the basis for highly time-resolved systems biology studies of metabolic switching in S. coelicolor A3(2). RESULTS: By a step-wise approach, cultivation conditions and two fully defined cultivation media were developed and evaluated using strain M145 of S. coelicolor A3(2), providing a high degree of cultivation reproducibility and enabling reliable studies of the effect of phosphate depletion and L-glutamate depletion on the metabolic transition to antibiotic production phase. Interestingly, both of the two carbon sources provided, D-glucose and L-glutamate, were found to be necessary in order to maintain high growth rates and prevent secondary metabolite production before nutrient depletion. Comparative analysis of batch cultivations with (i) both L-glutamate and D-glucose in excess, (ii) L-glutamate depletion and D-glucose in excess, (iii) L-glutamate as the sole source of carbon and (iv) D-glucose as the sole source of carbon, reveal a complex interplay of the two carbon sources in the bacterium's central carbon metabolism. CONCLUSIONS: The present study presents for the first time a dedicated cultivation strategy fulfilling the requirements for systems biology studies of metabolic switching in S. coelicolor A3(2). Key results from labelling and cultivation experiments on either or both of the two carbon sources provided indicate that in the presence of D-glucose, L-glutamate was the preferred carbon source, while D-glucose alone appeared incapable of maintaining culture growth, likely due to a metabolic bottleneck at the oxidation of pyruvate to acetyl-CoA.

Authors: A. Wentzel, P. Bruheim, A. Overby, O. M. Jakobsen, H. Sletta, W. A. Omara, D. A. Hodgson, T. E. Ellingsen

Date Published: 9th Jun 2012

Journal: BMC Syst Biol

Abstract (Expand)

Bacillus subtilis is known to accumulate large amounts of the compatible solute proline via de novo synthesis as a stress protectant when it faces high-salinity environments. We elucidated the genetic determinants required for the osmoadaptive proline production from the precursor glutamate. This proline biosynthesis route relies on the proJ-encoded γ-glutamyl kinase, the proA-encoded γ-glutamyl phosphate reductase, and the proH-encoded Δ1-pyrroline-5-caboxylate reductase. Disruption of the proHJ operon abolished osmoadaptive proline production and strongly impaired the ability of B. subtilis to cope with high-osmolarity growth conditions. Disruption of the proA gene also abolished osmoadaptive proline biosynthesis but caused, in contrast to the disruption of proHJ, proline auxotrophy. Northern blot analysis demonstrated that the transcription of the proHJ operon is osmotically inducible, whereas that of the proBA operon is not. Reporter gene fusion studies showed that proHJ expression is rapidly induced upon an osmotic upshift. Increased expression is maintained as long as the osmotic stimulus persists and is sensitively linked to the prevalent osmolarity of the growth medium. Primer extension analysis revealed the osmotically controlled proHJ promoter, a promoter that resembles typical SigA-type promoters of B. subtilis. Deletion analysis of the proHJ promoter region identified a 126-bp DNA segment carrying all sequences required in cis for osmoregulated transcription. Our data disclose the presence of ProA-interlinked anabolic and osmoadaptive proline biosynthetic routes in B. subtilis and demonstrate that the synthesis of the compatible solute proline is a central facet of the cellular defense to high-osmolarity surroundings for this soil bacterium.

Authors: Jeanette Brill, Tamara Hoffmann, Monika Bleisteiner, Erhard Bremer

Date Published: 22nd Jul 2011

Journal: J. Bacteriol.

Abstract (Expand)

Bacteria secrete numerous proteins into their environment for growth and survival under complex and ever-changing conditions. The highly different characteristics of secreted proteins pose major challenges to the cellular protein export machinery and, accordingly, different pathways have evolved. While the main secretion (Sec) pathway transports proteins in an unfolded state, the twin-arginine translocation (Tat) pathway transports folded proteins. To date, these pathways were believed to act in strictly independent ways. Here, we have employed proteogenomics to investigate the secretion mechanism of the esterase LipA of Bacillus subtilis, using a serendipitously obtained hyper-producing strain. While LipA is secreted Sec-dependently under standard conditions, hyper-produced LipA is secreted predominantly Tat-dependently via an unprecedented overflow mechanism. Two previously identified B. subtilis Tat substrates, PhoD and YwbN, require each a distinct Tat translocase for secretion. In contrast, hyper-produced LipA is transported by both Tat translocases of B. subtilis, showing that they have distinct but overlapping specificities. The identified overflow secretion mechanism for LipA focuses interest on the possibility that secretion pathway choice can be determined by environmental and intracellular conditions. This may provide an explanation for the previous observation that many Sec-dependently transported proteins have potential twin-arginine signal peptides for export via the Tat pathway.

Authors: Thijs R H M Kouwen, René van der Ploeg, Haike Antelmann, Michael Hecker, Georg Homuth, Ulrike Mäder, Jan Maarten Van Dijl

Date Published: 31st Jan 2009

Journal: Proteomics

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