Test of an anaerobic prototype reactor coupled with a filtration unit for production of VFAs.

The artificial ecosystem MELiSSA, supported by the European Space Agency is a closed loop system consisting of 5 compartments in which food, water and oxygen are produced out of organic waste. The first compartment is conceived as a thermophilic anaerobic membrane bioreactor liquefying organic waste into VFAs, ammonium and CO2 without methane. A 20 L reactor was assembled to demonstrate the selected design and process at prototype scale. We characterized system performance from start-up to steady state and evaluated process efficiencies with special attention drawn to the mass balances. An overall efficiency for organic matter biodegradation of 50% was achieved. The dry matter content was stabilized around 40-50 g L(-1) and VFA production around 5-6 g L(-1). The results were consistent for the considered substrate mixture and can also be considered relevant in a broader context, as a first processing step to produce building blocks for synthesis of primary energy vectors.

ß-Lactam and glycopeptide antibiotics: first and last line of defense?

Most infections are caused by bacteria, many of which are ever-evolving and resistant to nearly all available antibiotics. ß-Lactams and glycopeptides are used to combat these infections by inhibiting bacterial cell-wall synthesis. This mechanism remains an interesting target in the search for new antibiotics in light of failed genomic approaches and the limited input of major pharmaceutical companies. Several strategies have enriched the pipeline of bacterial cell-wall inhibitors; examples include combining screening strategies with lesser-explored microbial diversity, or reinventing known scaffolds based on structure-function relationships. Drugs developed using novel strategies will contribute to the arsenal in fight against the continued emergence of bacterial resistance.

Factors influencing cell fatty acid composition and A40926 antibiotic complex production in Nonomuraea sp. ATCC 39727.

A40926 is a glycopeptide antibiotic complex consisting of several structurally related factors. It is produced by fermentation of Nonomuraea sp. ATCC 39727 and the complex components differ in the structure of the fatty acid moiety linked to the aminoglucuronic acid unit. In previous work, we observed that the production of single factors in glycopeptide antibiotic complexes could be selectively enhanced by the addition of suitable precursors to the culture medium. In this contribution, we examine the effects of branched amino acid addition to fermentation of Nonomuraea sp. in a chemically defined minimal medium. The changes in the composition of cell fatty acids correlate to the fatty acid distribution within the A40926 complex in diverse cultivation conditions. Nonomuraea sp. prefers isobutyric, butyric and propionic acids as initiators of fatty acid biosynthesis. The relative amount of the produced fatty acids is significantly influenced by the availability of intermediates or final products from the amino acid catabolic pathways. Antibiotic complex composition closely reflects the cell fatty acid pattern, in agreement with the assumption that the antibiotic fatty acid moieties are synthesized by shortening the chain of cell fatty acids.

Biotransformations of lipoglycopeptides to obtain novel antibiotics.

The emergence of resistance among Gram-positive pathogens towards glycopeptide antibiotics has stimulated the research of second-generation molecules with improved activity and expanded antimicrobial spectrum. In this paper we investigate biotransformations as a way to generate novel teicoplanin- and A40926-like molecules. A range of commercial enzymes, fungi and actinomycetes were tested on A40926 and on its semi-synthetic derivatives (MDL 63,246 and dalbavancin). Oxidation of dalbavancin to MDL 63,246 was achieved by Nonomuraea sp. ATCC 39727 and Actinomadura parvosata ATCC 53463, while Actinoplanes sp. NRRL 3884, Actinoplanes missouriensis ATCC 23342 and Actinoplanes teichomyceticus ATCC 31121 deacylated MDL 63,246, dalbavancin and A40926. It is worth noting that the actinomycetes able to catalyze the deacylation of lipoglycopeptides are themselves producers of microbiologically active glycopeptides. Structurally related antibiotics (mideplanin and teicoplanin) were not transformed. Biotransformation conditions were optimised and scaled-up for the use of Actinoplanes sp. NRRL 3884 in the production of novel deacylated derivatives.

Valine influences production and complex composition of glycopeptide antibiotic A40926 in fermentations of Nonomuraea sp. ATCC 39727.

In actinomycetes the catabolism products of branched chain amino acids provide biosynthetic precursors for the formation of several lipid-containing antibiotics. We have determined in Nonomuraea sp. ATCC 39727 the effect of valine on production of glycopeptide antibiotic A40926, which is a complex of factors structurally differing in fatty acid moieties. Addition of valine to minimal medium increased A40926 production and modified complex composition towards a mono-component. Similar results were also obtained in a rich production medium.

Production and characterization of monochlorinated and dechlorinated A40926 derivatives.

Nonomuraea sp. ATCC 39727 is the producer of the A40926 complex of lipoglycopeptide antibiotics which contain chlorine atoms in amino acids 3 and 6 of the peptide backbone. Using a classical mutagenesis and selection approach we have isolated a Nonomuraea sp. ATCC 39727 mutant strain able to direct production towards new A40926 analogues dechloro-A40926 (DDC) lacking two chlorine atoms and the two monochloro-A40926 (MDC1 and MDC2) that are not produced fermenting the wild type strain. Dechlorinated A40926 derivatives were obtained in considerable amount in a standard fermentation process and were purified and chemically characterized. The dechlorinated A40926 derivatives DDC and MDC2 showed improved antimicrobial activity against coagulase negative staphylococci strains in respect to A40926 complex. Dechlorinated derivatives of the related antibiotic teicoplanin are also reported in the literature and are generally less active than the parental products.

Engineering of primary carbon metabolism for improved antibiotic production in Streptomyces lividans.

Deletions were made in Streptomyces lividans in either of two genes (zwf1 and zwf2) encoding isozymes of glucose-6-phosphate dehydrogenase, the first enzyme in the oxidative pentose phosphate pathway (PPP). Each mutation reduced the level of Zwf activity to approximately one-half that observed in the wild-type strain. When the mutants were transformed with multicopy plasmids carrying the pathway-specific transcriptional activator genes for either the actinorhodin (ACT) or undecylprodigiosin (RED) biosynthetic pathway, they produced higher levels of antibiotic than the corresponding wild-type control strains. The presumed lower flux of carbon through the PPP in each of the Deltazwf mutants may allow more efficient glucose utilization via glycolysis, resulting in higher levels of antibiotic production. This appears to occur without lowering the concentration of NADPH (the major biochemical product of the oxidative PPP activity) to a level that would limit antibiotic biosynthesis. Consistent with this hypothesis, deletion of the gene (devB) encoding the enzyme that catalyzes the next step in the oxidative PPP (6-phosphogluconolactonase) also resulted in increased antibiotic production. However, deletion of both zwf genes from the devB mutant resulted in reduced levels of ACT and RED production, suggesting that some of the NADPH made by the PPP is utilized, directly or indirectly, for antibiotic biosynthesis. Although applied here to the model antibiotics ACT and RED, such mutations may prove to be useful for improving the yield of commercially important secondary metabolites.