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Bjarne Gram Hansen - One of the best experts on this subject based on the ideXlab platform.

  • kinetically controlled drug resistance how Penicillium brevicompactum survives mycophenolic acid
    Journal of Biological Chemistry, 2011
    Co-Authors: Xin Sun, Bjarne Gram Hansen, Lizbeth Hedstrom
    Abstract:

    The filamentous fungus Penicillium brevicompactum produces the immunosuppressive drug mycophenolic acid (MPA), which is a potent inhibitor of eukaryotic IMP dehydrogenases (IMPDHs). IMPDH catalyzes the conversion of IMP to XMP via a covalent enzyme intermediate, E-XMP*; MPA inhibits by trapping E-XMP*. P. brevicompactum (Pb) contains two MPA-resistant IMPDHs, PbIMPDH-A and PbIMPDH-B, which are 17- and 103-fold more resistant to MPA than typically observed. Surprisingly, the active sites of these resistant enzymes are essentially identical to those of MPA-sensitive enzymes, so the mechanistic basis of resistance is not apparent. Here, we show that, unlike MPA-sensitive IMPDHs, formation of E-XMP* is rate-limiting for both PbIMPDH-A and PbIMPDH-B. Therefore, MPA resistance derives from the failure to accumulate the drug-sensitive intermediate.

  • a new class of imp dehydrogenase with a role in self resistance of mycophenolic acid producing fungi
    BMC Microbiology, 2011
    Co-Authors: Bjarne Gram Hansen, Hans Jasper Genee, Christian Schroder Kaas, Jakob Blaesbjerg Nielsen, Torsten Bak Regueira, Uffe Hasbro Mortensen, Jens Christian Frisvad, Kiran Raosaheb Patil
    Abstract:

    Background Many secondary metabolites produced by filamentous fungi have potent biological activities, to which the producer organism must be resistant. An example of pharmaceutical interest is mycophenolic acid (MPA), an immunosuppressant molecule produced by several Penicillium species. The target of MPA is inosine-5'-monophosphate dehydrogenase (IMPDH), which catalyses the rate limiting step in the synthesis of guanine nucleotides. The recent discovery of the MPA biosynthetic gene cluster from Penicillium brevicompactum revealed an extra copy of the IMPDH-encoding gene (mpaF) embedded within the cluster. This finding suggests that the key component of MPA self resistance is likely based on the IMPDH encoded by mpaF.

  • molecular basis for mycophenolic acid biosynthesis in Penicillium brevicompactum
    Applied and Environmental Microbiology, 2011
    Co-Authors: Torsten Bak Regueira, Bjarne Gram Hansen, Uffe Hasbro Mortensen, Kanchana Rueksomtawin Kildegaard, Christian Hertweck, Jens Nielsen
    Abstract:

    Mycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide synthase (PKS), MpaC, which we successfully characterized and identified as responsible for MPA production in Penicillium brevicompactum. mpaC resides in what most likely is a 25-kb gene cluster in the genome of Penicillium brevicompactum. The gene cluster was successfully localized by targeting putative resistance genes, in this case an additional copy of the gene encoding IMP dehydrogenase (IMPDH). We report the cloning, sequencing, and the functional characterization of the MPA biosynthesis gene cluster by deletion of the polyketide synthase gene mpaC of P. brevicompactum and bioinformatic analyses. As expected, the gene deletion completely abolished MPA production as well as production of several other metabolites derived from the MPA biosynthesis pathway of P. brevicompactum. Our work sets the stage for engineering the production of MPA and analogues through metabolic engineering.

  • versatile enzyme expression and characterization system for aspergillus nidulans with the Penicillium brevicompactum polyketide synthase gene from the mycophenolic acid gene cluster as a test case
    Applied and Environmental Microbiology, 2011
    Co-Authors: Bjarne Gram Hansen, Jakob Blaesbjerg Nielsen, Torsten Bak Regueira, Kiran Raosaheb Patil, Jens Nielsen, Bo Salomonsen, Morten Thrane Nielsen, Niels Hansen, Kristian Fog Nielsen, Uffe Hasbro Mortensen
    Abstract:

    Assigning functions to newly discovered genes constitutes one of the major challenges en route to fully exploiting the data becoming available from the genome sequencing initiatives. Heterologous expression in an appropriate host is central in functional genomics studies. In this context, filamentous fungi offer many advantages over bacterial and yeast systems. To facilitate the use of filamentous fungi in functional genomics, we present a versatile cloning system that allows a gene of interest to be expressed from a defined genomic location of Aspergillus nidulans. By a single USER cloning step, genes are easily inserted into a combined targeting-expression cassette ready for rapid integration and analysis. The system comprises a vector set that allows genes to be expressed either from the constitutive PgpdA promoter or from the inducible PalcA promoter. Moreover, by using the vector set, protein variants can easily be made and expressed from the same locus, which is mandatory for proper comparative analyses. Lastly, all individual elements of the vectors can easily be substituted for other similar elements, ensuring the flexibility of the system. We have demonstrated the potential of the system by transferring the 7,745-bp large mpaC gene from Penicillium brevicompactum to A. nidulans. In parallel, we produced defined mutant derivatives of mpaC, and the combined analysis of A. nidulans strains expressing mpaC or mutated mpaC genes unequivocally demonstrated that mpaC indeed encodes a polyketide synthase that produces the first intermediate in the production of the medically important immunosuppressant mycophenolic acid.

Angel Cantin - One of the best experts on this subject based on the ideXlab platform.

Pilar Moya - One of the best experts on this subject based on the ideXlab platform.

Uttam Chand Banerjee - One of the best experts on this subject based on the ideXlab platform.

  • machine learning modeling for ultrasonication mediated fermentation of Penicillium brevicompactum to enhance the release of mycophenolic acid
    Ultrasound in Medicine and Biology, 2021
    Co-Authors: Gopal Patel, Mahesh D Patil, Sujit Tangadpalliwar, Shivraj Hariram Nile, Prabha Garg, Guoyin Kai, Uttam Chand Banerjee
    Abstract:

    Abstract Described here is the modeling used to improve the mycophenolic acid (MPA) titer from Penicillium brevicompactum using central composite design and a comparatively newer, data-centric approach method k-nearest-neighbor algorithm. The two models for enhancing MPA production using P. brevicompactum were compared with respect to ultrasonic stimulation. During the ultrasonic treatment, we studied different independent factors such as ultrasound power, irradiation duration, treatment frequency and duty cycle to determine their ability to enhance the MPA titer value. The optimized factors such as a treatment time of 10 min (50% duty cycles) with a 12-h interlude at fixed ultrasonic power and frequency (200 W, 40 kHz) were used for ultrasonic treatment of a mycelial culture from the 2nd to 10th day of fermentation. Thus the production of MPA was improved 1.64-fold under the optimized sonication conditions compared with the non-sonicated batch fermentation (non-optimized conditions).

  • bioreactor studies of production of mycophenolic acid by Penicillium brevicompactum
    Biochemical Engineering Journal, 2018
    Co-Authors: Gopal Patel, Kush Biswas, Mahesh D Patil, Yusuf Chisti, Uttam Chand Banerjee
    Abstract:

    Abstract Batch and fed-batch fermentation processes for the production of mycophenolic acid (MPA) were optimized and compared. MPA was produced using the microfungus Penicillium brevicompactum. Different feeding strategies were assessed in fed-batch fermentations in a 14 L stirred bioreactor. A batch fermentation attained a maximum total (extracellular and intracellular) MPA concentration of 1.26 g/L after 288 h. The best fed-batch operation involving a constant-rate feeding of sorbitol (a feeding rate = 0.07 g/min, from 48 h to 240 h) and the pH controlled at 6, enhanced the total maximum MPA concentration to 3.26 g/L. The yield of MPA on substrate was 0.028 g/g, some 47% greater than in the aforementioned batch culture. The fermentation was highly aerobic. The fermentation temperature was 25 °C. Glucose could be used as an effective substrate instead of sorbitol, but the final total MPA concentration in a fed-batch fermentation using glucose was reduced to 2.72 g/L.

  • Production of Mycophenolic Acid by Penicillium brevicompactum Using Solid State Fermentation
    Applied Biochemistry and Biotechnology, 2017
    Co-Authors: Gopal Patel, Mahesh D Patil, Yusuf Chisti, Surbhi Soni, Uttam Chand Banerjee
    Abstract:

    Solid-state fermentation using the microfungus Penicillium brevicompactum for the production of mycophenolic acid is reported in this paper. Of the initial substrates tested (whole wheat, cracked wheat, long grain Basmati rice, and short grain Parmal rice), Parmal rice proved to be the best. Under initial conditions, using steamed Parmal rice with 80% ( w / w ) initial moisture content, a maximum mycophenolic acid concentration of 3.4 g/kg substrate was achieved in 12 days of fermentation at 25 °C. The above substrate was supplemented with the following additional nutrients (g/L packed substrate): glucose 40.0, peptone 54.0, KH_2PO_4 8.0, MgSO4⋅7H_2O 2.0, glycine 7.0, and methionine 1.65 (initial pH 5.0). A small amount of a specified trace element solution was also added. The final mycophenolic acid concentration was increased to nearly 4 g/kg substrate by replacing glucose with molasses. Replacing Parmal rice with rice bran as substrate further improved the mycophenolic acid production to nearly 4.5 g/kg substrate.

  • production of mycophenolic acid by Penicillium brevicompactum a comparison of two methods of optimization
    Biotechnology Reports, 2016
    Co-Authors: Gopal Patel, Mahesh D Patil, Yusuf Chisti, Surbhi Soni, Taresh P Khobragade, Uttam Chand Banerjee
    Abstract:

    Production of mycophenolic acid (MPA) by submerged fermentation using the microfungus Penicillium brevicompactum MTCC 8010 is reported here. Screening experiments were used to identify: the suitable media composition; the optimal initial pH; and the optimal incubation temperature to maximize the production of MPA in batch cultures. The initial concentrations of the selected sources of carbon (glucose), nitrogen (peptone) and the precursors (methionine, glycine) were then optimized by: (1) one-at-a-time variation of factors; and (2) a central composite design (CCD) of experiments, in a 12-day batch culture at an initial pH of 5.0, an incubation temperature of 25 °C, and an agitation speed of 200 rpm. The medium optimized using the one-at-a-time variation yielded a peak MPA titer of 1232 ± 90 mg/L. The medium optimized by the CCD method yielded a 40% higher MPA titer of 1737 ± 55 mg/L. The latter value was nearly 9-fold greater than the titer achieved prior to optimization.

Ulrich Kuck - One of the best experts on this subject based on the ideXlab platform.

  • molecular analysis of mating type loci from the mycophenolic acid producer Penicillium brevicompactum phylogeny and mat protein characterization suggest a cryptic sexual life cycle
    Fungal Biology, 2020
    Co-Authors: Yasaman Mahmoudjanlou, Tim A Dahlmann, Ulrich Kuck
    Abstract:

    Abstract The mycophenolic acid producing ascomycete Penicillium brevicompactum is considered to be an anamorphic (asexual) species, for which a sexual cycle was never observed. However, since recent reports of otherwise asexually propagating filamentous fungi have demonstrated a sexual cycle controlled by mating type loci, we carried out a molecular analysis of mating type loci from P. brevicompactum. Using data from extensive DNA sequencing analysis, we determined the mating type loci from 22 strains derived from various type culture collections. We found 8 strains carrying a MAT1-1 locus encoding a 362 amino acid alpha domain transcription factor. The other 14 possessed a MAT1-2 locus encoding a 298 amino acid HMG domain transcription factor. cDNA analysis confirmed that both mating type loci are transcriptionally expressed. The karyotype of six selected strains, determined using contour-clamped homogeneous electric field (CHEF) electrophoresis, demonstrated distinct differences in size and numbers of chromosomes between the strains investigated. Interestingly, our phylogenetic survey of 72 strains from 11 different Penicillium species revealed that MAT genes serve as excellent molecular markers to determine phylogenetic relationships among species closely related to P. brevicompactum. Based on our sequencing results, we constructed transformation vectors for site-specific deletion of mating type loci from two selected strains of opposite mating type. Complementation strains were constructed containing both the mating type locus deletion cassette and a MAT-egfp fusion gene. These strains were used for comparative phenotypic analyses between strains containing or lacking the mating type gene. Whereas all MAT1-2 strains were indistinguishable, the MAT1-1 and MAT1-1-1 deletion strains differed distinctly. The MAT1-1-1 deletion strain produced more conidiospores on solid media, but smaller pellets in liquid media. This is probably the consequence of fewer conidial germ tubes than with the wild type mating type strain. Finally, we showed that the MAT-EGPF fusion protein is localized to the nuclei and detectable in protein samples by Western analysis. Together, our results suggest that the asexually propagating fungus P. brevicompactum might be a heterothallic species with a cryptic sexual life cycle.

  • construction of a codon adapted nourseotricin resistance marker gene for efficient targeted gene deletion in the mycophenolic acid producer Penicillium brevicompactum
    Journal of Fungi, 2019
    Co-Authors: Yasaman Mahmoudjanlou, Birgit Hoff, Ulrich Kuck
    Abstract:

    Penicillium brevicompactum is a filamentous ascomycete used in the pharmaceutical industry to produce mycophenolic acid, an immunosuppressant agent. To extend options for genetic engineering of this fungus, we have tested two resistance markers that have not previously been applied to P. brevicompactum. Although a generally available phleomycin resistance marker (ble) was successfully used in DNA-mediated transformation experiments, we were not able to use a commonly applicable nourseothricin resistance cassette (nat1). To circumvent this failure, we constructed a new nat gene, considering the codon bias for P. brevicompactum. We then used this modified nat gene in subsequent transformation experiments for the targeted disruption of two nuclear genes, MAT1-2-1 and flbA. For MAT1-2-1, we obtained deletion strains with a frequency of about 10%. In the case of flbA, the frequency was about 4%, and this disruption strain also showed reduced conidiospore formation. To confirm the deletion, we used ble to reintroduce the wild-type genes. This step restored the wild-type phenotype in the flbA deletion strain, which had a sporulation defect. The successful transformation system described here substantially extends options for genetically manipulating the biotechnologically relevant fungus P. brevicompactum.