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17. Additional file 1 of Metabolic engineering of Deinococcus radiodurans for pinene production from glycerol

Author

Helalat, Seyed Hossein, Jers, Carsten, Bebahani, Mandana, Mohabatkar, Hassan, and Mijakovic, Ivan

Abstract

Additional file 1: Table S1. List of oligos used for vector construction and real-time PCR. Figure S1. The sequence of the pinene synthase-encoding gene codon-optimized for D. radiodurans. Figure S2. Pinene degradation or consumption by D. radiodurans in 120 h. D. radiodurans was grown in a medium supplemented with 20 mg/L of pinene isomers and residual pinene was quantified by GC-FID after 120 h. Un-inoculated pinene supplemented medium was used as the control. Figure S3. Effect of using different concentrations of dodecane (10% and 20%) a in the rich and b the minimal glycerol medium and various agitation speed (70, 100, 180, and 250 rpm) on the pinene production by D. radiodurans ∆dr0862 P-I-D. Figure S4. GC-FID chromatograms. a Alpha-pinene standard, b beta-pinene standard, c beta-pinene production by D. radiodurans, and d alpha- and beta-pinene production by E. coli. Figure S5. Pinene synthase expression in E. coli EcpsB by different concentrations of IPTG. SDS-PAGE 4 h after induction.

Published
2021
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28. Phosphorylation of [i]Bacillus subtilis[/i] gene regulator AbrB modulates its DNA-binding properties

Author

Kobir, Ahasanul, Poncet-Mouturat, Sandrine, Bidnenko, Vladimir, Delumeau, Olivier, Jers, Carsten, Zouhir, Samira, Grenha, Rosa, Nessler, Sylvie, Noirot, Philippe, Mijakovic, Ivan, MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, UPR3082 Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Department of Chemical and Biological Engineering, Systems and Synthetic Biology, Chalmers University of Technology [Göteborg], and Institut National de la Recherche Agronomique

Subjects
DNA-Binding Proteins, Bacterial Proteins, [SDV]Life Sciences [q-bio], fungi, Gene Expression Regulation, Bacterial, Phosphorylation, Bacillus subtilis, Transcription Factors
Abstract

AbrB is a global gene regulator involved in transition phase phenomena in Bacillus subtilis. It participates in a complex regulatory network governing the expression of stationary-phase functions. AbrB was previously found to be phosphorylated on serine 86 located close to its C-terminal oligomerization domain. Here we report that AbrB can be phosphorylated by three B. subtilis serine/threonine kinases expressed during the transition and stationary phase: PrkC, PrkD and YabT. Our in vitro findings suggest that AbrB phosphorylation impedes its DNA binding and abolishes binding cooperativity. In vivo we established that a phospho-mimetic mutation abrB S86D leads to a significant loss of AbrB control over several key target functions: exoprotease production, competence development and sporulation. A wider transcriptome analysis of abrB S86D and S86A mutant strains revealed deregulation of a large number of target genes. We therefore propose that AbrB phosphorylation serves as an additional input for fine-tuning the activity of this ambiactive gene regulator.

Published
2014

39. Automated N-glycan profiling of a mutant Trypanosoma rangeli sialidase expressed in Pichia pastoris, using tandem mass spectrometry and bioinformatics.

Author

Haiying Li, Rasmussen, Morten I., Larsen, Martin R., Yao Guo, Jers, Carsten, Palmisano, Giuseppe, Mikkelsen, Jørn D., and Kirpekar, Finn

Subjects
GLYCANS, NEURAMINIDASE, TRYPANOSOMA rangeli, PICHIA pastoris, TANDEM mass spectrometry, BIOINFORMATICS, PROTEIN expression, FUNCTIONAL analysis
Abstract

A mutant Trypanosoma rangeli sialidase, Tr7, expressed in Pichia pastoris, exhibits significant transsialidase activity, and has been used for analytical-scale production of sialylated human milk oligosaccharides. Mass spectrometry-based site-specific N-glycoprofiling of Tr7 showed that heterogeneous high-mannose type N-glycans were present at all the five potential N-linked glycosites. N-linked glycans in Tr7 were predominantly neutral oligosaccharides with compositions Man8-16GlcNAc2, but also mono- and di-phosphorylated oligosaccharides in the forms of Man9-15P1GlcNAc2 and Man9-14P2GlcNAc2, respectively. Some phosphorylated N-linked glycans further contained an additional HexNAc, which has not previously been reported in P. pastorisexpressed proteins. We compiled a method pipeline that combined hydrophilic interaction liquid chromatography enrichment of glycopeptides, high accuracy mass spectrometry and automated interpretation of the mass spectra with in-house developed "MassAI" software, which proved efficient in glycan site microheterogeneity analysis. Functional analysis showed that the deglycosylated Tr7 retained more than 90% of both the sialidase and trans-sialidase activities relative to the glycosylated Tr7. [ABSTRACT FROM AUTHOR]

Published
2015
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44. Phosphorylation of B acillus subtilis gene regulator AbrB modulates its DNA-binding properties.

Author

Kobir, Ahasanul, Poncet, Sandrine, Bidnenko, Vladimir, Delumeau, Olivier, Jers, Carsten, Zouhir, Samira, Grenha, Rosa, Nessler, Sylvie, Noirot, Phillipe, and Mijakovic, Ivan

Subjects
BACILLUS subtilis genetics, PHOSPHORYLATION, DNA-binding proteins, GENETIC regulation, BACTERIAL mutation, SERINE/THREONINE kinase regulation, BACTERIA
Abstract

AbrB is a global gene regulator involved in transition phase phenomena in B acillus subtilis. It participates in a complex regulatory network governing the expression of stationary-phase functions. AbrB was previously found to be phosphorylated on serine 86 located close to its C-terminal oligomerization domain. Here we report that AbrB can be phosphorylated by three B . subtilis serine/threonine kinases expressed during the transition and stationary phase: PrkC, PrkD and YabT. Our in vitro findings suggest that AbrB phosphorylation impedes its DNA binding and abolishes binding cooperativity. In vivo we established that a phospho-mimetic mutation abrB S86 D leads to a significant loss of AbrB control over several key target functions: exoprotease production, competence development and sporulation. A wider transcriptome analysis of abrB S86 D and S86 A mutant strains revealed deregulation of a large number of target genes. We therefore propose that AbrB phosphorylation serves as an additional input for fine-tuning the activity of this ambiactive gene regulator. [ABSTRACT FROM AUTHOR]

Published
2014
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45. Rational Design of a New Trypanosoma rangeli Trans-Sialidase for Efficient Sialylation of Glycans.

Author

Jers, Carsten, Michalak, Malwina, Larsen, Dorte M., Kepp, Kasper P., Li, Haiying, Guo, Yao, Kirpekar, Finn, Meyer, Anne S., and Mikkelsen, Jørn D.

Subjects
*TRYPANOSOMA rangeli, *GENETIC engineering, *GLYCANS, *NEURAMINIDASE, *AMINO acid analysis, *GENE expression
Abstract

This paper reports rational engineering of Trypanosoma rangeli sialidase to develop an effective enzyme for a potentially important type of reactivity: production of sialylated prebiotic glycans. The Trypanosoma cruzi trans-sialidase and the homologous T. rangeli sialidase has previously been used to investigate the structural requirements for trans-sialidase activity. We observed that the T. cruzi trans-sialidase has a seven-amino-acid motif (197–203) at the border of the substrate binding cleft. The motif differs substantially in chemical properties and substitution probability from the homologous sialidase, and we hypothesised that this motif is important for trans-sialidase activity. The 197–203 motif is strongly positively charged with a marked change in hydrogen bond donor capacity as compared to the sialidase. To investigate the role of this motif, we expressed and characterised a T. rangeli sialidase mutant, Tr13. Conditions for efficient trans-sialylation were determined, and Tr13's acceptor specificity demonstrated promiscuity with respect to the acceptor molecule enabling sialylation of glycans containing terminal galactose and glucose and even monomers of glucose and fucose. Sialic acid is important in association with human milk oligosaccharides, and Tr13 was shown to sialylate a number of established and potential prebiotics. Initial evaluation of prebiotic potential using pure cultures demonstrated, albeit not selectively, growth of Bifidobacteria. Since the 197–203 motif stands out in the native trans-sialidase, is markedly different from the wild-type sialidase compared to previous mutants, and is shown here to confer efficient and broad trans-sialidase activity, we suggest that this motif can serve as a framework for future optimization of trans-sialylation towards prebiotic production. [ABSTRACT FROM AUTHOR]

Published
2014
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46. Quantitative Phosphoproteome Analysis of Bacillus subtilisReveals Novel Substrates of the Kinase PrkC and Phosphatase PrpC*

Author

Ravikumar, Vaishnavi, Shi, Lei, Krug, Karsten, Derouiche, Abderahmane, Jers, Carsten, Cousin, Charlotte, Kobir, Ahasanul, Mijakovic, Ivan, and Macek, Boris

Abstract

Reversible protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) residues plays a critical role in regulation of vital processes in the cell. Despite of considerable progress in our understanding of the role of this modification in bacterial physiology, the dynamics of protein phosphorylation during bacterial growth has rarely been systematically addressed. In addition, little is known about in vivosubstrates of bacterial Ser/Thr/Tyr kinases and phosphatases. An excellent candidate to study these questions is the Gram-positive bacterium Bacillus subtilis,one of the most intensively investigated bacterial model organism with both research and industrial applications. Here we employed gel-free phosphoproteomics combined with SILAC labeling and high resolution mass spectrometry to study the proteome and phosphoproteome dynamics during the batch growth of B. subtilis. We measured the dynamics of 1666 proteins and 64 phosphorylation sites in five distinct phases of growth. Enzymes of the central carbon metabolism and components of the translation machinery appear to be highly phosphorylated in the stationary phase, coinciding with stronger expression of Ser/Thr kinases. We further used the SILAC workflow to identify novel putative substrates of the Ser/Thr kinase PrkC and the phosphatase PrpC during stationary phase. The overall number of putative substrates was low, pointing to a high kinase and phosphatase specificity. One of the phosphorylation sites affected by both, PrkC and PrpC, was the Ser281 on the oxidoreductase YkwC. We showed that PrkC phosphorylates and PrpC dephosphorylates YkwC in vitroand that phosphorylation at Ser281 abolishes the oxidoreductase activity of YkwC in vitroand in vivo. Our results present the most detailed phosphoproteomic analysis of B. subtilisgrowth to date and provide the first global in vivoscreen of PrkC and PrpC substrates.

Published
2014
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