微生物学技术

The transcriptome has shown tremendous potential for the comprehensive investigation of gene expression profiles and transcriptional levels in comparative biology, the identification of regulatory mechanism of transcriptional regulators, and the evaluation of target ...

The continuous emergence of multidrug-resistant bacterial pathogens is a major problem in public health. Many mechanisms may be involved in such resistance in Staphylococcus aureus. Increasing data have shown that S. aureus can invade different types of host cells, which may contribu ...

Here, we describe a solid-phase microextraction-gas chromatography/mass spectrometry (SPME-GC/MS) analytical approach that identifies and analyzes volatile compounds in the headspace above a live fungal culture. This approach is a sensitive, solvent-free, robust techni ...

Ergot alkaloids are indole derivatives with diverse structures and biological activities. This chapter describes the procedure from fungal cultivation to purified ergot alkaloids, as exemplified by fumigaclavine A in Penicillium commune. Furthermore, useful notes for work ...

Non-ribosomal peptide synthetases (NRPSs) are important enzymes of production machinery for natural products including clinically used antibiotics, antifungal, and anticancer agents. NRPS products are usually further modified by tailoring enzymes, resulting in the form ...

Filamentous fungi have long been recognized to be a rich source of secondary metabolites with potential medicinal applications. The recent genomic sequencing of several Aspergillus species has revealed that many secondary metabolite gene clusters are apparently silent under s ...

The FERMEX (Fermentation Extract) program was a highly successful source of microbial natural product molecules for pharmaceutical lead discovery. The program was based on the observation that solid fermentations of fungi generally exhibited more complex metabolite profiles ...

Methods for manipulating and fermenting microorganisms in multi-well plates offer unlimited possibilities for high-throughput parallel experimentation. Furthermore, bar-coded data tracking and downstream processing with modern liquid handling equipment reduce ...

Growth media and incubation conditions have a very strong influence of secondary metabolite production. There is no consensus on which media are the optimal for metabolite production, but a series of useful and effective media and incubation conditions have been listed here. Chemically ...

The accelerating pace of microbial genomics is sparking a renaissance in the field of natural products research. Researchers can now get a preview of the organism’s secondary metabolome by analyzing its genomic sequence. Combined with other -omics data, this approach may provide a cost-e ...

Fungi produce an astonishing variety of secondary metabolites, some of which belong to the most toxic compounds in the living world. However, the benefits of secondary metabolites for fungi are often obscure. Here we describe a method that will aid in the analysis of fungal gene expression upon in ...

The hollow fiber assay (HFA) is a drug discovery tool to aid investigators in the prioritization of lead compounds identified by in vitro testing for further development in animal models of disease. In the HFA, cells are cultured in hollow fibers containing pores of a diameter (500 kDa) large enough f ...

High-throughput sequencing (HTS) has quickly become a valuable tool for comparative genetics and genomics and is now regularly carried out in laboratories that are not connected to large sequencing centers. Here we describe an updated version of our protocol for constructing single- and ...

Recent studies conducted in our laboratory demonstrate that Aspergillus parasiticus synthesizes and stores aflatoxin in transport vesicles and endosomes. Proteomics data suggest that enzymes involved in the synthesis of other secondary metabolites as well as enzymes invol ...

Understanding in vivo protein–protein interactions is critical to dissect precise functions of the regulatory proteins of fungal secondary metabolites. As many fungi differentially produce a diverse array of secondary metabolites during their lifecycle, it is important to un ...

A crucial part of optimization of metabolically engineered organisms is producing balanced levels of pathway proteins. Typically, protein levels are monitored by Western blot analysis; however, application to multiple enzyme pathways can be difficult without unique antibodi ...

Chromatin immunoprecipitation (ChIP) is used to map the interaction between proteins and DNA at a specific genomic locus in the living cell. The protein–DNA complexes are stabilized already in vivo by reversible crosslinking and the DNA is sheared by sonication or enzymatic digestion in ...

For many fungi the number of known secondary metabolites is surprisingly small compared to the �astonishingly large number of terpene cyclase, polyketide synthase (PKS), and non-ribosomal peptide synthetase (NRPS) secondary metabolite gene clusters found in their genomes. Corr ...

Fungal molecular biology has benefited from the enormous advances in understanding protein–protein interactions in prokaryotic or eukaryotic organisms of the past decade. Tandem affinity purification (TAP) allows the enrichment of native protein complexes from cell extra ...

With the completion of genomes of various Aspergillus species, large-scale genome-wide expression studies can be carried out. Genomics, however, is more powerful and efficient when combined with genetics. A multi-copy-based gain-of-function screen is a complementary method to lo ...