Biotransformation of Trinitrotoluene (TNT) by Newly Isolated Slight Halophilic Bacteria

Microbiology - Current study reports isolation of newly isolated slight halophilic bacteria which can utilize 2,4,6-trinitrotoluene (TNT) as a sole nitrogen source, leading to its detoxification....     

The Penicillium Chrysogenum Extracellular Proteome. Conversion from a Food-rotting Strain to a Versatile Cell Factory for White Biotechnology

The filamentous fungus Penicillium chrysogenum is well-known by its ability to synthesize β-lactam antibiotics as well as other secondary metabolites. Like other filamentous fungi, this microorganism is an excellent host for secretion of extracellular proteins because of the high capacity of its protein secretion machinery. In this work, we have characterized the extracellular proteome reference map of P. chrysogenum Wisconsin 54–1255 by two-dimensional gel electrophoresis. This method allowed the correct identification of 279 spots by peptide mass fingerprinting and tandem MS. These 279 spots included 328 correctly identified proteins, which corresponded to 131 different proteins and their isoforms. One hundred and two proteins out of 131 were predicted to contain either classical or nonclassical secretion signal peptide sequences, providing evidence of the authentic extracellular location of these proteins. Proteins with higher representation in the extracellular proteome were those involved in plant cell wall degradation (polygalacturonase, pectate lyase, and glucan 1,3-β-glucosidase), utilization of nutrients (extracellular acid phosphatases and 6-hydroxy-d-nicotine oxidase), and stress response (catalase R). This filamentous fungus also secretes enzymes specially relevant for food industry, such as sulfydryl oxidase, dihydroxy-acid dehydratase, or glucoamylase. The identification of several antigens in the extracellular proteome also highlights the importance of this microorganism as one of the main indoor allergens. Comparison of the extracellular proteome among three strains of P. chrysogenum, the wild-type NRRL 1951, the Wis 54–1255 (an improved, moderate penicillin producer), and the AS-P-78 (a penicillin high-producer), provided important insights to consider improved strains of this filamentous fungus as versatile cell-factories of interest, beyond antibiotic production, for other aspects of white biotechnology.Filamentous fungi have an extraordinary ability to secrete proteins, secondary metabolites, and organic acids to the culture medium. The secreted proteins play important roles in nutrition, substrate colonization, or pathogenicity (1). This high secretory capacity has made filamentous fungi attractive for the commercial production of extracellular proteins (2), especially for the food and beverage industries (3), which have been using the compounds secreted by filamentous fungi for decades. Examples are provided by Aspergillus oryzae, which has been important for the production of traditional fermented foods and beverages in Japan and is used in modern biotechnology because of its ability to secrete large amounts of proteins (4) or Aspergillus niger, which has been widely used in biotechnology for the production of organic acids, food ingredients, and industrial enzymes (5). Because A. oryzae, A. niger, and Penicillium chrysogenum belong to the same fungal family, the latter microorganism might be considered of interest for the secretion of extracellular proteins.Although the understanding of the molecular basis of the secretion process in filamentous fungi is still limited (1), it is generally accepted that the secretion pathway in these microorganisms does not differ greatly from that present in yeasts and higher eukaryotes and protein secretion is believed to occur mainly at hyphal tips (6). The classical secretory pathway of proteins is driven by a canonical N-terminal signal peptide. These proteins enter the endoplasmic reticulum, where they are properly folded and modified (glycosylation, phosphorylation, etc.) and subsequently reach the Golgi compartment packed in transport vesicles. In this compartment, proteins can undergo further additional modifications such as glycosylation and peptide processing. Following this step, proteins are packed in secretory vesicles directed to the plasma membrane for secretion, or targeted to the vacuole either to become resident proteins or to undergo proteolytic degradation (7). In addition to the classical endoplasmic reticulum-Golgi pathway, it has been suggested that various kinds of mechanistically distinct nonclassical export routes may exist (8, 9). Cytoplasmic, nuclear and signal-peptide-containing proteins have been shown to reach the cell surface by nonconventional transport pathways (10). In yeasts, other mechanisms of secretion, which drive proteins lacking the signal peptide outside the plasma membrane, have also been described (11).P. chrysogenum is a filamentous fungus well-known by its ability to synthesize β-lactam antibiotics such as benzylpenicillin and isopenicillin N (12). Because the isolation of the wild-type strain NRRL 1951 from an infected cantaloupe in Peoria, Illinois in 1943 (13), this microorganism has undergone artificial selection by mutagenesis during industrial strain improvement programs, which gave rise to the improved-producing Wisconsin 54–1255 strain (hereafter named Wis 54-1255) (14). This strain became a laboratory model strain and was used for the genome sequencing project (15) and the intracellular proteome reference map (16). P. chrysogenum Wis 54–1255 was the ancestor of penicillin high-producing mutants, such as the AS-P-78 strain developed by Antibióticos S.A (León, Spain). The mutagenesis processes undergone by the P. chrysogenum strains during the industrial selection have introduced several important modifications in their metabolic networks (16).The recent advances in the Proteomics tools and the availability of genome sequences, has allowed an analysis of the secretomes of a few filamentous fungi, but the available information is still scarce (17–19). However, because of the availability of several fungal genomes and diverse prediction programs for secretory proteins, an integrated platform for annotation of fungal secretomes (Fungal Secretome Database) has been established and implemented in a web-based database (20). This database has been proposed as an integrated environment for the study of secretory proteins in the fungal kingdom.In order to fully characterize P. chrysogenum and to establish how the modifications acquired during the industrial strain improvement programs affected the wild type plant pathogenicity, analysis of the secreted proteins present in the culture broths was carried out. Using two-dimensional gel electrophoresis (2-DE)1 gels coupled to peptide mass fingerprint (PMF) and tandem MS we describe here for the first time the extracellular proteome of P. chrysogenum and the differences found in secreted protein among the wild type and two improved strains of this microorganism. Results reveal the nutritional versatility of this filamentous fungus and its potential interest for other biotechnological purposes different from antibiotic production, because nonpenicillin producer strains have been previously developed (21) that lack the penicillin biosynthesis genes (22) and can be used for other biotechnological uses.     

Taxonomy and chemically semi-defined media for the analysis of the tacrolimus producer ‘Streptomyces tsukubaensis’

‘Streptomyces tsukubaensis’ was the first tacrolimus producer strain identified. Although it has been included in the Streptomyces genus, its taxonomic position has not been rigorously determined. By using a polyphasic approach, we have established that the tacrolimus producer strain ‘S. tsukubaensis’ NRRL 18488 represents a unique species in the Streptomyces genus, which is phylogenetically distant from other subsequently described producers. This fact means a horizontal transference of the tacrolimus-producing gene cluster. Physiology, nutrient requirement, and molecular genetics analyses of tacrolimus biosynthesis in ‘S. tsukubaensis’ necessitate chemically defined or semi-defined media, which work as a jigsaw puzzle and allow for pieces (nutrients) exchange. To date, studies related to ‘S. tsukubaensis’ have been mainly focused in the improvement of tacrolimus production using complex industrial fermentation media, which difficulty allows testing of tacrolimus overproduction enhancers or inhibitors because of the presence of non‐defined substances. In the present work, two semi-defined media were developed in order to study the main factors involved in tacrolimus production in ‘S. tsukubaensis’.     

Characterisation of a ��-butyrolactone receptor of Streptomyces tacrolimicus: effect on sporulation and tacrolimus biosynthesis

Streptomyces tacrolimicus (ATCC 55098) was reported to produce the immunosuppressant tacrolimus. The wild-type strain sporulates sparsely and produces very low levels of this immunosuppressant. The lack of genetic knowledge of this strain has hampered strain improvement. In this work, we have cloned the gene encoding a γ-butyrolactone receptor protein (Gbr). The gbr gene is linked to two genes encoding two subunits of the dihydroxyacetone kinase, putatively involved in the biosynthesis of the dihydroxyacetone phosphate precursor of γ-butyrolactone but is not flanked by γ-butyrolactone synthetase genes. The Gbr protein was overexpressed in Escherichia coli and purified. Electrophoretic mobility shift assays showed that Gbr binds to a specific autoregulatory element sequence located 338 bp upstream of the gbr gene, indicating that its expression is self-regulated. The deletion mutant Δgbr showed a very early and intense sporulation in two different media. A phenotype similar to that of the wild-type strain was restored by complementation of the Δgbr mutant with a wild-type gbr allele. Duplication of the gbr gene resulted in a slower sporulation. The Δgbr mutant produced much lower amount (32%) of tacrolimus quantified by high performance liquid chromatography. This analysis, using an optimised system, allowed the resolution of tacrolimus from ascomycin and other contaminant metabolites. Our results indicate that the Gbr protein regulates negatively the sporulation and positively the production of tacrolimus.     

Draft genome of Streptomyces tsukubaensis NRRL 18488, the producer of the clinically important immunosuppressant tacrolimus (FK506)

The macrocyclic polyketide tacrolimus (FK506) is a potent immunosuppressant that prevents T-cell proliferation produced solely by Streptomyces species. We report here the first draft genome sequence of a true FK506 producer, Streptomyces tsukubaensis NRRL 18488, the first tacrolimus-producing strain that was isolated and that contains the full tacrolimus biosynthesis gene cluster.