Structural Variation among Wild and Industrial Strains of Penicillium chrysogenum

Strain selection and strain improvement are the first, and arguably most important, steps in the industrial production of biological compounds by microorganisms. While traditional methods of mutagenesis and selection have been effective in improving production of compounds at a commercial scale, the genetic changes underpinning the altered phenotypes have remained largely unclear. We utilized high-throughput Illumina short read sequencing of a wild Penicillium chrysogenum strain in order to make whole genome comparisons to a sequenced improved strain (WIS 54–1255). We developed an assembly-free method of identifying chromosomal rearrangements and validated the in silico predictions with a PCR-based assay and Sanger sequencing. Despite many rounds of mutagen treatment and artificial selection, WIS 54–1255 differs from its wild progenitor at only one of the identified rearrangements. We suggest that natural variants predisposed for high penicillin production were instrumental in the success of WIS 54–1255 as an industrial strain. In addition to finding a previously published inversion in the penicillin biosynthesis cluster, we located several genes related to penicillin production associated with these rearrangements. By comparing the configuration of rearrangement events among several historically important strains known to be high penicillin producers to a collection of recently isolated wild strains, we suggest that wild strains with rearrangements similar to those in known high penicillin producers may be viable candidates for further improvement efforts.     

A Branched Biosynthetic Pathway Is Involved in Production of Roquefortine and Related Compounds in Penicillium chrysogenum

Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopiperazine (DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO) which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway.     

Caenorhabditis elegans: A Simple Nematode Infection Model for Penicillium marneffei

Penicillium marneffei, one of the most important thermal dimorphic fungi, is a severe threat to the life of immunocompromised patients. However, the pathogenic mechanisms of P. marneffei remain largely unknown. In this work, we developed a model host by using nematode Caenorhabditis elegans to investigate the virulence of P. marneffei. Using two P. marneffei clinical isolate strains 570 and 486, we revealed that in both liquid and solid media, the ingestion of live P. marneffei was lethal to C. elegans (P<0.001). Meanwhile, our results showed that the strain 570, which can produce red pigment, had stronger pathogenicity in C. elegans than the strain 486, which can’t produce red pigment (P<0.001). Microscopy showed the formation of red pigment and hyphae within C. elegans after incubation with P. marneffei for 4 h, which are supposed to be two contributors in nematodes killing. In addition, we used C. elegans as an in vivo model to evaluate different antifungal agents against P. marneffei, and found that antifungal agents including amphotericin B, terbinafine, fluconazole, itraconazole and voriconazole successfully prolonged the survival of nematodesinfected by P. marneffei. Overall, this alternative model host can provide us an easy tool to study the virulence of P. marneffei and screen antifungal agents.     

Genome-scale model for Clostridium acetobutylicum: Part I. Metabolic network resolution and analysis

A genome-scale metabolic network reconstruction for Clostridium acetobutylicum (ATCC 824) was carried out using a new semi-automated reverse engineering algorithm. The network consists of 422 intracellular metabolites involved in 552 reactions and includes 80 membrane transport reactions. The metabolic network illustrates the reliance of clostridia on the urea cycle, intracellular L-glutamate solute pools, and the acetylornithine transaminase for amino acid biosynthesis from the 2-oxoglutarate precursor. The semi-automated reverse engineering algorithm identified discrepancies in reaction network databases that are major obstacles for fully automated network-building algorithms. The proposed semi-automated approach allowed for the conservation of unique clostridial metabolic pathways, such as an incomplete TCA cycle. A thermodynamic analysis was used to determine the physiological conditions under which proposed pathways (e.g., reverse partial TCA cycle and reverse arginine biosynthesis pathway) are feasible. The reconstructed metabolic network was used to create a genome-scale model that correctly characterized the butyrate kinase knock-out and the asolventogenic M5 pSOL1 megaplasmid degenerate strains. Systematic gene knock-out simulations were performed to identify a set of genes encoding clostridial enzymes essential for growth in silico.     

基于Methylovorus sp.J1-1基因组尺度代谢网络优化吡咯喹啉醌合成

通过构建Methylovorus sp.J1-1基因组尺度代谢网络模型(genome scale of metabolic network model,GSMM),发掘能够提升吡咯喹啉醌(pyrroloquinoline quinone,PQQ)产量的发酵策略和相关靶基因.基于其注释的基因组和生化信息,构建J1-1的G...     

In Vitro Metabolic and Mitogenic Signaling of Insulin Glargine and Its Metabolites

Background

Insulin glargine (Lantus®) is a long-acting basal insulin analog that demonstrates effective day-long glycemic control and a lower incidence of hypoglycemia than NPH insulin. After subcutaneous injection insulin glargine is partly converted into the two main metabolites M1 ([Gly^A21]insulin) and M2 ([Gly^A21,des-Thr^B30]insulin). The aim of this study was to characterize the glargine metabolites in vitro with regard to their insulin receptor (IR) and IGF-1 receptor (IGF1R) binding and signaling properties as well as their metabolic and mitogenic activities.

Methods

The affinity of human insulin, insulin glargine and its metabolites to the IR isoforms A and B or IGF1R was analyzed in a competitive binding assay using SPA technology. Receptor autophosphorylation activities were studied via In-Cell Western in CHO and MEF cells overexpressing human IR-A and IR-B or IGF1R, respectively. The metabolic response of the insulins was studied as stimulation of lipid synthesis using primary rat adipocytes. Thymidine incorporation in Saos-2 cells was used to characterize the mitogenic activity.

Conclusions

The binding of insulin glargine and its metabolites M1 and M2 to the IR were similar and correlated well with their corresponding autophosphorylation and metabolic activities in vitro. No differences were found towards the two IR isoforms A or B. Insulin glargine showed a higher affinity for IGF1R than insulin, resulting in a lower EC₅₀ value for autophosphorylation of the receptor and a more potent stimulation of thymidine incorporation in Saos-2 cells. In contrast, the metabolites M1 and M2 were significantly less active in binding to and activation of the IGF1R and their mitogenicity in Saos-2 cells was equal to human insulin. These findings strongly support the idea that insulin glargine metabolites contribute with the same potency as insulin glargine to blood glucose control but lead to significantly reduced growth-promoting activity.     

Metabolic fluxes for nutritional flexibility of Mycobacterium tuberculosis

The co‐catabolism of multiple host‐derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady‐state chemostat system. We demonstrate that Mtb efficiently co‐metabolises either cholesterol or glycerol, in combination with two‐carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.     

The pbrB Gene Encodes a Laccase Required for DHN-Melanin Synthesis in Conidia of Talaromyces (Penicillium) marneffei

Talaromyces marneffei (Basionym: Penicillium marneffei) is a significant opportunistic fungal pathogen in patients infected with human immunodeficiency virus in Southeast Asia. T. marneffei cells have been shown to become melanized in vivo. Melanins are pigment biopolymers which act as a non-specific protectant against various stressors and which play an important role during virulence in fungi. The synthesis of the two most commonly found melanins in fungi, the eumelanin DOPA-melanin and the allomelanin DHN-melanin, requires the action of laccase enzymes. The T. marneffei genome encodes a number of laccases and this study describes the characterization of one of these, pbrB, during growth and development. A strain carrying a PbrB-GFP fusion shows that pbrB is expressed at high levels during asexual development (conidiation) but not in cells growing vegetatively. The pbrB gene is required for the synthesis of DHN-melanin in conidia and when deleted results in brown pigmented conidia, in contrast to the green conidia of the wild type.     

Expanding the Species and Chemical Diversity of Penicillium Section Cinnamopurpurea

A set of isolates very similar to or potentially conspecific with an unidentified Penicillium isolate NRRL 735, was assembled using a BLAST search of ITS similarity among described (GenBank) and undescribed Penicillium isolates in our laboratories. DNA was amplified from six loci of the assembled isolates and sequenced. Two species in section Cinnamopurpurea are self-compatible sexual species, but the asexual species had polymorphic loci suggestive of sexual reproduction and variation in conidium size suggestive of ploidy level differences typical of heterothallism. Accordingly we use genealogical concordance analysis, a technique valid only in heterothallic organisms, for putatively asexual species. Seven new species were revealed in the analysis and are described here. Extrolite analysis showed that two of the new species, P. colei and P. monsserratidens produce the mycotoxin citreoviridin that has demonstrated pharmacological activity against human lung tumors. These isolates could provide leads in pharmaceutical research.     

Development of a Markerless Genetic Exchange System for Desulfovibrio vulgaris Hildenborough and Its Use in Generating a Strain with Increased Transformation Efficiency

In recent years, the genetic manipulation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has seen enormous progress. In spite of this progress, the current marker exchange deletion method does not allow for easy selection of multiple sequential gene deletions in a single strain because of the limited number of selectable markers available in D. vulgaris. To broaden the repertoire of genetic tools for manipulation, an in-frame, markerless deletion system has been developed. The counterselectable marker that makes this deletion system possible is the pyrimidine salvage enzyme, uracil phosphoribosyltransferase, encoded by upp. In wild-type D. vulgaris, growth was shown to be inhibited by the toxic pyrimidine analog 5-fluorouracil (5-FU), whereas a mutant bearing a deletion of the upp gene was resistant to 5-FU. When a plasmid containing the wild-type upp gene expressed constitutively from the aph(3′)-II promoter (promoter for the kanamycin resistance gene in Tn5) was introduced into the upp deletion strain, sensitivity to 5-FU was restored. This observation allowed us to develop a two-step integration and excision strategy for the deletion of genes of interest. Since this in-frame deletion strategy does not retain an antibiotic cassette, multiple deletions can be generated in a single strain without the accumulation of genes conferring antibiotic resistances. We used this strategy to generate a deletion strain lacking the endonuclease (hsdR, DVU1703) of a type I restriction-modification system that we designated JW7035. The transformation efficiency of the JW7035 strain was found to be 100 to 1,000 times greater than that of the wild-type strain when stable plasmids were introduced via electroporation.The anaerobic sulfate-reducing bacteria (SRB) are found in a remarkable variety of habitats. These bacteria have received attention recently because they have a potential role in toxic metal bioremediation (23, 26). To fully understand the potential benefits and to maximize opportunities for successful manipulation of the SRB, it would be useful to create deletions in critically important genes. Several activities of particular interest are represented by multiple isozymes, suggesting that compensation may occur upon elimination of one or more of these genes. To fully elucidate alternative pathways, genetic approaches allowing the construction of multiple mutations are needed. The genetic manipulation of the SRB Desulfovibrio vulgaris Hildenborough has seen significant improvements in recent years (reviewed in reference 3). Chloramphenicol and kanamycin marker exchange mutagenesis methods have been developed (2, 10). Although gene deletions can be constructed, the necessary retention of antibiotic resistance limits sequential deletions, since each deletion would require an additional antibiotic cassette. To eliminate the necessity of marker retention, an in-frame markerless deletion system has been developed.A two-step method for marker exchange/deletion that used the counterselectable marker sacB (13) was used by Fu and Voordouw (10) to generate the first deletion by marker exchange in D. vulgaris. The sacB gene from Bacillus subtilis encodes levansucrase and confers sensitivity to sucrose in many gram-negative bacteria (7, 32, 35), including D. vulgaris (8, 10, 15, 18, 19, 21). In the first step of the process, a suicide plasmid carrying DNA regions from up- and downstream of a target gene flanking a chloramphenicol resistance (Cm^r) cassette was introduced into D. vulgaris by conjugation and single recombinants were selected as Cm^r colonies (10). After confirmation of the integration of this plasmid, the double-recombination event was selected on medium containing chloramphenicol and sucrose. This method, with some variation, has been used to make several mutants by the Voordouw group (8, 10, 15, 18, 19, 21). One unexpected complication was the observation that 50% of sucrose resistant colonies were due to events other than the removal of the sacB gene and plasmid through a second recombination as desired (11). Also, sensitivity to sucrose is apparently strongly affected by medium composition, initial culture density, and the time of exposure (11). This method involved a large time investment, but it ultimately resulted in a marker exchange mutant (Cm^r) and established the effectiveness of a two-step recombination process in D. vulgaris.Among alternative counterselectable markers are the purine and pyrimidine salvage enzymes, phosphoribosyl transferases (PRTases). These enzymes allow the recycling of free bases from internal or environmental sources, as well as the incorporation of base analogs into nucleoside monophosphates. Importantly, the incorporation of base analogs can be lethal and are the reason these nucleotide salvage pathways have been widely used as counterselectable markers for gene knockout systems in bacteria, archaea, and eukaryotes (4, 5, 8, 9, 12, 16, 22, 24, 27, 29, 34). Specifically, the incorporation of the pyrimidine analog 5-fluorouracil (5-FU) is lethal in a number of bacteria (8, 16, 22). Mutants whose genes encoding the pertinent PRTases have been deleted are resistant to the toxic base analogs (4, 5, 8, 9, 12, 16, 22, 24, 27, 29, 34). Reintroduction of these genes restores sensitivity. In order to utilize the genes for PRTases as counterselectable markers, a deletion of the endogenous PRTase gene must be created in the host strain. We have previously shown that wild-type D. vulgaris is extremely sensitive to low levels of 5-FU, as little as 0.1 μg/ml (3). In the present study, we deleted the upp gene (DVU1025) encoding the putative uracil phosphoribosyl transferase in D. vulgaris creating strain JW710 and showed that it was resistant to 5-FU. When the upp gene was reintroduced into JW710 (Δupp), it restored sensitivity to wild-type levels of 5-FU. These phenotypic observations indicate that the loss of the upp provides a selectable marker for a two-step integration and excision strategy for the deletion of target genes without a residual marker exchange. A second advantage of using this markerless method is the facile ability to generate in-frame deletions, eliminating potential polarity.To test the effectiveness of using the upp as a counterselectable marker in D. vulgaris, we deleted the gene encoding the endonuclease of a type I restriction-modification system, hsdR (DVU1703), and the downstream conserved hypothetical gene (CHP; DVU1702), creating strain JW7035 [Δupp Δ(hsdR-CHP)]. The type I restriction-modification system was targeted for deletion in hopes of increasing the transformation efficiency of D. vulgaris and facilitating the construction of future deletions. As anticipated, electroporation experiments with stable plasmids revealed an improvement in transformation efficiency for stable plasmids compared to wild-type D. vulgaris. Finally, Gateway Technology (Invitrogen) was applied to generate a destination vector (pMO727) containing the constitutively expressed wild-type upp gene. This vector will expedite the process of creating the required suicide deletion vectors for future markerless deletions.     

Sex in Cheese: Evidence for Sexuality in the Fungus Penicillium roqueforti

Although most eukaryotes reproduce sexually at some moment of their life cycle, as much as a fifth of fungal species were thought to reproduce exclusively asexually. Nevertheless, recent studies have revealed the occurrence of sex in some of these supposedly asexual species. For industrially relevant fungi, for which inoculums are produced by clonal-subcultures since decades, the potentiality for sex is of great interest for strain improvement strategies. Here, we investigated the sexual capability of the fungus Penicillium roqueforti, used as starter for blue cheese production. We present indirect evidence suggesting that recombination could be occurring in this species. The screening of a large sample of strains isolated from diverse substrates throughout the world revealed the existence of individuals of both mating types, even in the very same cheese. The MAT genes, involved in fungal sexual compatibility, appeared to evolve under purifying selection, suggesting that they are still functional. The examination of the recently sequenced genome of the FM 164 cheese strain enabled the identification of the most important genes known to be involved in meiosis, which were found to be highly conserved. Linkage disequilibria were not significant among three of the six marker pairs and 11 out of the 16 possible allelic combinations were found in the dataset. Finally, the detection of signatures of repeat induced point mutations (RIP) in repeated sequences and transposable elements reinforces the conclusion that P. roqueforti underwent more or less recent sex events. In this species of high industrial importance, the induction of a sexual cycle would open the possibility of generating new genotypes that would be extremely useful to diversify cheese products.