No abstract

This review presents an update on the current knowledge of the secondary metabolite potential of the major fungal species used in industrial biotechnology, i.e., Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei. These species have a long history of safe use for enzyme production. Like most microorganisms that exist in a challenging environment in nature, these fungi can produce a large variety and number of secondary metabolites. Many of these compounds present several properties that make them attractive for different industrial and medical applications. A description of all known secondary metabolites produced by these species is presented here. Mycotoxins are a very limited group of secondary metabolites that can be produced by fungi and that pose health hazards in humans and other vertebrates when ingested in small amounts. Some mycotoxins are species-specific. Here, we present scientific basis for (1) the definition of mycotoxins including an update on their toxicity and (2) the clarity on misclassification of species and their mycotoxin potential reported in literature, e.g., A. oryzae has been wrongly reported as an aflatoxin producer, due to misclassification of Aspergillus flavus strains. It is therefore of paramount importance to accurately describe the mycotoxins that can potentially be produced by a fungal species that is to be used as a production organism and to ensure that production strains are not capable of producing mycotoxins during enzyme production. This review is intended as a reference paper for authorities, companies, and researchers dealing with secondary metabolite assessment, risk evaluation for food or feed enzyme production, or considerations on the use of these species as production hosts.

Members of the ‘Bacillus subtilis group’ include some of the most commercially important bacteria, used for the production of a wide range of industrial enzymes and fine biochemicals. Increasingly, group members have been developed for use as animal feed enhancers and antifungal biocontrol agents. The group has long been recognised to produce a range of secondary metabolites and, despite their long history of safe usage, this has resulted in an increased focus on their safety. Traditional methods used to detect the production of secondary metabolites and other potentially harmful compounds have relied on phenotypic tests. Such approaches are time consuming and, in some cases, lack specificity. Nowadays, accessibility to genome data and associated bioinformatical tools provides a powerful means for identifying gene clusters associated with the synthesis of secondary metabolites. This review focuses primarily on well-characterised strains of B. subtilis and B. licheniformis and their synthesis of non-ribosomally synthesised peptides and polyketides. Where known, the activities and toxicities of their secondary metabolites are discussed, together with the limitations of assays currently used to assess their toxicity. Finally, the regulatory framework under which such strains are authorised for use in the production of food and feed enzymes is also reviewed.

SummaryIn a previous study, we described the use of transposon Tn917-LTV1 for identi®cation of environmentally regulated promoters in Lactococcus lactis. Here, we report the molecular analysis of one of these promoters, P170, that is upregulated at low pH during the transition to stationary phase. The minimal DNA region required for both promoter activity and pH regulation was mapped to a 51 bp fragment located 7 bp upstream of the transcriptional start site. This fragment lacked the consensus À35 promoter region, but it contained an`extended' À10 promoter region. When a 28 bp segment, containing the consensus À35 region and 22 bp upstream of this in a constitutive promoter, was replaced with the corresponding sequence of P170, the hybrid promoter became regulated by pH and growth phase. This demonstrates that the P170 segment contains a cis-acting sequence involved in the control of promoter regulation. Transcriptional analysis showed that P170 is responsible for the transcription of a monocistronic gene orfX encoding a polypeptide homologous to a hypothetical protein from Bacillus subtilis. Analysis of total RNA from L. lactis grown at constant pH con®rmed that transcription from P170 was induced between pH 6.5 and pH 6.0, but only when the culture entered stationary phase. Deletion analysis and chemical mutagenesis of P170 de®ned a speci®c region within the untranslated mRNA leader that is able to modulate the expression level directed by the P170 promoter. Deletion of a 72 bp HaeIII fragment from this leader region resulted in a 150-to 200-fold increase in the level of gene expression, without affecting the regulation. The functionality was con®rmed by introducing this modulating element downstream of other lactococcal promoters.

hDDPI (human dipeptidyl peptidase I) is a lysosomal cysteine protease involved in zymogen activation of granule-associated proteases, including granzymes A and B from cytotoxic T-lymphocytes and natural killer cells, cathepsin G and neutrophil elastase, and mast cell tryptase and chymase. In the present paper, we provide the first crystal structure of an hDPPI-inhibitor complex. The inhibitor Gly-Phe-CHN2 (Gly-Phe-diazomethane) was co-crystallized with hDPPI and the structure was determined at 2.0 A (1 A=0.1 nm) resolution. The structure of the native enzyme was also determined to 2.05 A resolution to resolve apparent discrepancies between the complex structure and the previously published structure of the native enzyme. The new structure of the native enzyme is, within the experimental error, identical with the structure of the enzyme-inhibitor complex presented here. The inhibitor interacts with three subunits of hDPPI, and is covalently bound to Cys234 at the active site. The interaction between the totally conserved Asp1 of hDPPI and the ammonium group of the inhibitor forms an essential interaction that mimics enzyme-substrate interactions. The structure of the inhibitor complex provides an explanation of the substrate specificity of hDPPI, and gives a background for the design of new inhibitors.