Hexaene Derivatives of Nystatin Produced as a Result of an Induced Rearrangement within the nysC Polyketide Synthase Gene in S. noursei ATCC 11455

AbstractGenetic manipulation of the polyketide synthase (PKS) gene nysC involved in the biosynthesis of the tetraene antifungal antibiotic nystatin yielded a recombinant strain producing hexaene nystatin derivatives. Analysis of one such compound, S48HX, by LC-MS/MS suggested that it comprises a 36-membered macrolactone ring completely decorated by the post-PKS modification enzymes. Further characterization by bioassay has shown that S48HX exhibits antifungal activity. Genetic analysis of the hexaene-producing mutant revealed an in-frame deletion within the nysC gene via recombination between two homologous ketoreductase domain-encoding sequences. Apparently, this event resulted in the elimination of one complete module from NysC PKS, subsequently leading to the production of the nystatin derivative with a contracted macrolactone ring. These results represent the first example of manipulation of a PKS gene for the biosynthesis of a polyene antibiotic

Structural and mutational characterization of the catalytic A-module of the mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii

Alginate is a family of linear copolymers of (1→4)-linked β-d-mannuronic acid and its C-5 epimer α-l-guluronic acid. The polymer is first produced as polymannuronic acid and the guluronic acid residues are then introduced at the polymer level by mannuronan C-5-epimerases. The structure of the catalytic A-module of the Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 has been determined by x-ray crystallography at 2.1-Å resolution. AlgE4A folds into a right-handed parallel β-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The β-helix is composed of four parallel β-sheets, comprising 12 complete turns, and has an amphipathic α-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp(152), an amino acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr(149), His(154), and Asp(178) as being essential for activity. Tyr(149) probably acts as the proton acceptor, whereas His(154) is the proton donor in the epimerization reaction

NMR assignments of 1H, 13C and 15N resonances of the C-terminal subunit from Azotobacter vinelandii mannuronan C5-epimerase 6 (AlgE6R3)

The 19.9 kDa C-terminal module (R3) from Azotobacter vinelandii mannronan C5-epimerase AlgE6 has been 13C, 15N isotopically labelled and recombinantly expressed. We report here the 1H, 13C, 15N resonance assignment of AlgE6R3

Meeting report : 1st international functional metagenomics workshop May 7–8, 2012, St. Jacobs, Ontario, Canada

This report summarizes the events of the 1st International Functional Metagenomics Workshop. The workshop was held on May 7 and 8 in St. Jacobs, Ontario, Canada and was focused on building a core international functional metagenomics community, exploring strategic research areas, and identifying opportunities for future collaboration and funding. The workshop was initiated by researchers at the University of Waterloo with support from the Ontario Genomics Institute (OGI), Natural Sciences and Engineering Research Council of Canada (NSERC) and the University of Waterloo

Special Issue: Recombinant Protein Expression in Microorganisms

Microorganisms are widely used in industrial biotechnology as cell factories for the sustainable production of a wide range of compounds and chemicals [...

Strong stimulation of recombinant protein production in <it>Escherichia coli </it>by combining stimulatory control elements in an expression cassette

<p>Abstract</p> <p>Background</p> <p>The XylS/<it>Pm</it> expression system has been used to produce recombinant proteins at industrial levels in <it>Escherichia coli</it>. Activation of transcription from the <it>Pm</it> promoter takes place in the presence of benzoic acid or derivatives of it. Previous mutagenesis studies resulted in identification of several variants of the expression control elements <it>xylS</it> (X), <it>Pm</it> (P) and the 5'-untranslated region (U) that individually gave rise to strongly stimulated expression. The goal of this study was to test if combination of such stimulatory mutations in the same expression vectors would lead to further increase of expression levels.</p> <p>Results</p> <p>We combined X, P and U variants that were originally identified due to their ability to strongly stimulate expression of the reporter gene <it>bla</it> (resistance to penicillin). Combination of optimized elements stimulated <it>bla</it> expression up to 75-fold (X, P and U combined) relative to the wild-type system, while accumulated transcript levels increased about 50-fold. This is much more than for the elements individually. We also tested combination of the variant elements on two other and unrelated genes, <it>celB</it> (encoding phosphoglucomutase) and the human growth factor gene <it>gm-csf</it>. Protein production from these genes is much more efficient than from <it>bla</it> in the wild-type system, but expression was still significantly stimulated by the combination of X, P and U variants, although not to the same extent as for <it>bla</it>.</p> <p>We also integrated a single copy of the expression cassette with each gene into the <it>E. coli</it> chromosome and found that the expression level from this single copy was higher for <it>bla</it> than for the wild-type plasmid system, while it was lower for <it>celB</it> and <it>gm-csf</it>.</p> <p>Conclusion</p> <p>Our results show that combination of stimulatory expression control elements can be used to further increase production of different proteins in <it>E. coli</it>. For one reporter gene (<it>bla</it>) this allowed for more protein production from a single gene copy integrated on the chromosome, compared to the wild-type plasmid system. The approach described here should in principle be applicable for improvement of any expression cassette.</p