The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.

Kluyveromyces lactis is both scientifically and biotechnologically one of the most important non-Saccharomyces yeasts. Its biotechnological significance builds on its history of safe use in the food industry and its well-known ability to produce enzymes like lactase and bovine chymosin on an industrial scale. In this article, we review the various strains, genetic techniques and molecular tools currently available for the use of K. lactis as a host for protein expression. Additionally, we present data illustrating the recent use of proteomics studies to identify cellular bottlenecks that impede heterologous protein expression.

We compared the response of Saccharomyces cerevisiae to carbon (glucose) and nitrogen (ammonia) limitation in chemostat cultivation at the proteome level. Protein levels were differentially quantified using unlabeled and 15 N metabolically labeled yeast cultures. A total of 928 proteins covering a wide range of isoelectric points, molecular weights and subcellular localizations were identified. Stringent statistical analysis identified 51 proteins upregulated in response to glucose limitation and 51 upregulated in response to ammonia limitation. Under glucose limitation, typical glucose-repressed genes encoding proteins involved in alternative carbon source utilization, fatty acids b-oxidation and oxidative phosphorylation displayed an increased protein level. Proteins upregulated in response to nitrogen limitation were mostly involved in scavenging of alternative nitrogen sources and protein degradation. Comparison of transcript and protein levels clearly showed that upregulation in response to glucose limitation was mainly transcriptionally controlled, whereas upregulation in response to nitrogen limitation was essentially controlled at the posttranscriptional level by increased translational efficiency and/or decreased protein degradation. These observations underline the need for multilevel analysis in yeast systems biology.

The use of chemostat culturing enables investigation of steady-state physiological characteristics and adaptations to nutrient-limited growth, while all other relevant growth conditions are kept constant. We examined and compared the proteomic response of wild-type Saccharomyces cerevisiae CEN.PK113-7D to growth in aerobic chemostat cultures limited for carbon sources being either glucose or ethanol. To obtain a global overview of changes in the proteome, we performed triplicate analyses using two-dimensional gel electrophoresis and identified proteins of interest using MS. Relative quantities of about 400 proteins were obtained and analyzed statistically to determine which protein steady-state expression levels changed significantly under glucose-or ethanollimited conditions. Interestingly, only enzymes involved in central carbon metabolism showed a significant change in steady-state expression, whereas expression was only detected in one of both carbon source-limiting conditions for 15 of these enzymes. Side effects that were previously reported for batch cultivation conditions, such as responses to continuous variation of specific growth rate, to carbon-catabolite repression, and to accumulation of toxic substrates, were not observed. Moreover, by comparing our proteome data with corresponding mRNA data, we were able to unravel which processes in the central carbon metabolism were regulated at the level of the proteome, and which processes at the level of transcriptome. Importantly, we show here that the combined approach of chemostat cultivation and comprehensive proteome analysis allowed us to study the primary effect of single limiting conditions on the yeast proteome. Molecular & Cellular Proteomics 4:1-11, 2005. Two-dimensional (2D)1 gel electrophoresis is a powerful tool to visualize hundreds of proteins at a time, which in combination with MS leads to their identification. This technology has been applied to the yeast Saccharomyces cerevisiae for the large-scale identification of more than 400 proteins, resulting in yeast reference maps (1-7). Other S. cerevisiae studies used 2D gel electrophoresis to obtain an overview of global changes in the yeast proteome as function of stimuli such as cadmium (8), lithium (9), H 2 O 2 (10), or sorbic acid (11).Most of these differential proteome studies on S. cerevisiae were performed on cells cultured in batch mode, i.e. in shake flasks or in reactors. Batch cultivation makes use of a closed system, in which all nutrients are in excess at the start of the cultivation. In terms of microbial physiology, such batch cultivation is relatively poorly controlled, because the composition of the growth medium and consequently the growth rate changes continuously. The yeast cells take up nutrients from the media while metabolites are excreted in the culture system, and their growth arrests when one of the nutrients is depleted or when too many toxic substrates are accumulated. The high concentration of carbon source in the culture, which is essential for this type of cultu...

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The effect of annealing temperature on molecular interactions at the interface of polymer laminates is reported. Depth profiling has been carried out by using confocal Raman microspectroscopy to study poly(acrylonitrile)/poly(vinyl alcohol) (PAN/PVOH) and poly(acrylonitrile)/poly(acrylic acid) (PAN/PAA) laminates. The laminates have been annealed at 65, 75, and 90 °C. It is demonstrated that the degree of hydrogen-bonding interaction between the nitrile and alcohol groups near the interfacial region changes between laminates annealed at different temperatures. Increasing the annealing temperature up to near the glass transition temperature, Tg, of both polymers facilitates hydrogen bonding. However, above Tg, as a result of molecular flexibility, weakening of such interactions begins. Due to the lower molecular weight of PAA, and thus greater mobility in comparison with PVOH, the interfacial region of a PAN/PAA laminate is broader than that of a PAN/PVOH laminate. Hydrogen-bonding interaction between PAN and PAA was not observed. This result is rationalized by the hypothesis that, as a result of intramolecular interaction between the carbonyl and hydroxyl groups of PAA, formation of intermolecular hydrogen bonding has been hindered. In addition, the variations in the full width at half-height (FWHH) of the v(C≡N) band of a PAN–PVOH blend has been mapped by using the same technique.

To monitor the structural integrity of therapeutic proteins, hydrogen–deuterium exchange mass spectrometry (HDX-MS) is increasingly utilized in the pharmaceutical industry. The successful outcome of HDX-MS analyses depends on the sample preparation conditions, which involve the rapid digestion of proteins at 0 °C and pH 2.5. Very few proteases are able to withstand such harsh conditions, with pepsin being the best-known exception, even though its activity is also strongly reduced at 0 °C. Here, we evaluate the usage of a prolyl endopeptidase from Aspergillus niger (An-PEP) for HDX-MS. What makes this protease very attractive is that it cleaves preferentially the hardest to digest amino acid, proline. To our surprise, and in contrast to previous reports, An-PEP activity was found optimal around pH 2.5 and could be further enhanced by urea up to 40%. Under typical HDX-MS conditions and using small amounts of enzyme, An-PEP generated an equivalent number of peptides as pepsin, as exemplified by using the two model systems tetrameric human hemoglobin (Hb) and human IgG4. Interestingly, because An-PEP peptides are shorter than pepsin-generated peptides, higher sequence resolution could be achieved, especially for Pro-containing protein regions in the alpha subunit of Hb, revealing new protected Hb regions that were not observed with pepsin. Due to its Pro-preference and resistance to low pH, we conclude that An-PEP is an archetype enzyme for HDX-MS, highly complementary to pepsin, and especially promising for structural studies on Pro-rich proteins or proteins containing Pro-rich binding domains involved in cellular signaling.

In an attempt to find the best approach for the mass spectrometric analysis of the whole range of lipopolysaccharide (LPS) structures from Klebsiella pneumoniae ssp. pneumoniae rough strain R20 (O1-:K20-), various methods of LPS preparation were applied and the products were analyzed using a range of mass spectrometric techniques. The most productive approach proved to be the removal of lipid A by mild acid hydrolysis and the study of the core oligosaccharide structures using nanoelectrospray time-of-flight mass spectrometry (TOF-MS) in combination with collision-induced dissociation tandem mass spectrometry. This procedure is very sensitive, but results in the generation of a reducing 3-deoxy-D-manno-oct-2-ulopyranosonic acid residue (Kdo) that is susceptible to the formation of artifacts, which give rise to pseudomolecular ions 18, 46, and 88 Da below the pseudomolecular ion for the unmodified species. Alternatively, matrix-assisted laser desorption/ionization TOF-MS combined with post-source decay can be used to study the de-O-acylated LPS preparation and especially to identify those residues bearing phosphate groups and the residues involved in the linkage between the core and lipid A. In addition to the five LPS core structures defined using NMR spectroscopy by Süsskind et al., several extra related LPS structure were identified. Larger LPS species were observed, which surprisingly do not represent species containing longer versions of the novel Klebsiella heptoglycan, but instead are species having the defined core and heptoglycan extended with up to three extra hexuronic acid and one or two extra hexose residues.