Kinetically controlled drug resistance: how Penicillium brevicompactum survives mycophenolic acid

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 10(3)-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.     

霉酚酸产生菌原生质体转基因方法的建立

摘要:【目的】建立霉酚酸产生菌短密青霉菌的遗传转化体系。【方法】以腐草霉素抗性为选择标记,利用聚乙二醇介导原生质体融合,进行外源基因转化。【结果】聚乙二醇介导的短密青霉菌原生质体转化效率为每微克DNA 2－3个转化子;转化子的PCR检测结果显示外源基因已经整合到短密青霉菌基因组中,转化子抗性稳定。【结论】霉酚酸产生菌短密青霉菌转基因体系的建立为该菌进行分子生物学研究以及基因工程育种奠定了基础。     

Occurrence and biosynthesis of asperphenamate in solid cultures of Penicillium brevicompactum

The ester of N-benzoylphenylalanine and N-benzoylphenylalaninol, asperphenamate, was isolated from solid cultures of Penicillium brevicompactum. Isotope from l-[U-¹⁴C] phenylalanine was well incorporated into both benzoyl groups and into the phenylalanine and phenylalaninol moieties. Isotope from [U-¹⁴C]benzoic acid was also well incorporated into asperphenamate.     

Mycophenolic acid production by Penicillium brevicompactum in two media

Penicillium brevicompactum produces mycophenolic acid as it grows vegetatively, not only on a simple medium where growth is slow but also on a richer medium where growth is less restricted. The implications of this finding on the association of fungal secondary metabolism with the idiophase in liquid and solid culture are discussed.     

Labelled acetone and levulinic acid are formed when [14C]acetate is being converted to mycophenolic acid in Penicillium brevicompactum

Evidence is presented that is compatible with the hypothesis that the farnesyl unit involved in the biosynthesis of mycophenolic acid in Penicillium brevicompactum is degraded by two successive oxidative cleavages at the double bonds distal from the phthalide nucleus. Acetone and levulinic acid are two non-aromatic degradation products.     

Effect of culture medium components on the biosynthesis of acid ribonuclease by Penicillium brevicompactum cultures]

The effect of different ingredients of the nutrient medium on the synthesis of acid ribonuclease by the culture Penicillium brevi-compactum was studied. The synthesis of RNase reached maximum on the medium containing glucose, pepton, soya bean flour, ammonium sulphate. An excessive content of phosphate in the medium decreased the RNase synthesis. An optimal medium for the synthesis of the enzymes was developed and recommended.     

Molecular basis for cytokinin biosynthesis

Cytokinins (CKs) are a group of phytohormones that play a crucial role in the regulation of plant growth and development. Identification of the enzymes and the corresponding genes that are involved in CK metabolism allowed us to understand how plants synthesize CKs and adjust CK activity to optimal levels. A major accomplishment toward these goals was the identification of genes for the first enzyme in the CK biosynthetic pathway, adenosine phosphate-isopentenyltransferase (IPT). In Arabidopsis thaliana and Agrobacterium tumefaciens, detailed analyses of IPTs were conducted through not only enzymatic characterization but also molecular structural approaches. These studies revealed the molecular basis for the Agrobacterium-origin of IPT used for the efficient biosynthesis of trans-zeatin that promotes tumorigenesis in host plants. Another landmark in CK research was the identification of CYP735A as an enzyme that converts iP-nucleotide to tZ-nucleotide. Furthermore, the identification of a CK-activating enzyme, LOG, which catalyzes a novel activation pathway, is a remarkable recent achievement in CK research. Collectively, these advances have revealed the complexity of the entire metabolic scheme for CK biosynthesis.     

Amino acid sequence and S-S bonds of Penicillium brevicompactum guanyl-specific ribonuclease

The primary structure of Penicillium brevicompactum guanyl-specific RNase was determined. The enzyme consists of 102 amino acid residues, Mr 10801. The 4 cysteine residues of the RNase are linked in pairs by disulfide bonds: Cys2-Cys10, Cys6-Cys101. P. brevicompactum RNase structure is similar to RNase T1; the degree of homology is 66%.     

Screening and identification of a Penicillium brevicompactum strain isolated from the fruiting body of Inonotus obliquus and the fermentation production of mycophenolic acid

Mycophenolic acid (MPA) is a fungal metabolite with a variety of biological activities and widely applied in clinical practices. We herein aimed to isolate a new Penicillium brevicompactum strain from the fruiting body of Inonotus obliquus to improve the production of MPA. The fruiting body of Inonotus obliquus was used to isolate P. brevicompactum strains. Identification of the P. brevicompactum strain was performed by sequencing and phylogenetic tree analysis. HPLC assay was conducted to identify the production of MPA. Submerged liquid fermentation and bi-directional fermentation were applied to improve the yield of MPA. The candidates of the P. brevicompactum strain were isolated and screened from the fruiting body of Inonotus obliquus collected from Changbai mountains in China. Based on sequencing and phylogenetic tree analysis of 18S rDNA-ITS, the strain MC-4 was finally identified as P. brevicompactum. And HPLC assay indicated that the isolated P. brevicompactum strain could produce MPA in metabolic products. The optimized conditions of submerged liquid fermentation were as follows: 100 g/L Chinese yam in a liquid PDB medium, pH 6, fermentation temperature of 24 °C, shaker speed of 130 r/min, and fermentation time of 6 days. The maximum value of MPA production was 1.415 g/L after submerged liquid fermentation. Furthermore, the yield of MPA could be significantly increased to 1.537 g/L after bi-directional fermentation with the extractive from fructus Swietenia macrophylla (FSM). We demonstrated that a Penicillium brevicompactum strain isolated from the fruiting body of Inonotus obliquus can be used to improve the production of MPA by submerged liquid fermentation and bi-directional fermentation. This would provide a novel approach for more efficient and safer production of MPA.     

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

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.     

Role of cinnamate in benzoate production in Penicillium brevicompactum

Radioisotope feeding experiments with solid cultures of Penicillium brevicompactum demonstrate that cinnamate is an intermediate in the conversion of l-phenylalanine to benzoate. The first enzyme in this pathway, phenylalanine ammonia-lyase, has been purified 62-fold from surface liquid cultures.     

Production of mycophenolic acid by fungi of the genus Penicillium link

Out of 36 strains of fungi of the genus Penicillium, some of which were isolated from ancient permafrost soils, 14 strains synthesized mycophenolic acid (MPA). Maximal (over 500 mg/l) accumulation of MPA in culture liquid was observed in P. brevicompactum strains (VKM F-457, VKM F-477, and VKM F-1150). This was the first study to detect MPA in representatives of the species P. rugulosum; in three strains of this species (VKM FW-665, VKM FW-717, and VKM FW-733), the level of MPA accumulation exceeded 300 mg/l. The time course of the synthesis of MPA by the P. rugulosum strain VKM FW-733 was studied. It was shown that the synthesis of this metabolite was dramatically intensified at the stationary growth phase (ten days).     

Molecular basis for biosynthesis and accumulation of polyhydroxyalkanoic acids in bacteria

Abstract The current knowledge on the structure and on the organization of polyhydroxyalkanoic acid (PHA)-biosynthetic genes from a wide range of different bacteria, which rely on different pathways for biosynthesis of this storage polyesters, is provided. Molecular data will be shown for genes of Alcaligenes eutrophus , purple non-sulfur bacteria, such as Rhodospirillum rubrum , purple sulfur bacteria, such as Chromatium vinosum , pseudomonads belonging to rRNA homology group I, such as Pseudomonas aeruginosa, Methylobacterium extorquens , and for the Gram-positive bacterium Rhodococcus ruber . Three different types of PHA synthases can be distinguished with respect to their substrate specificity and structure. Strategies for the cloning of PHA synthase structural genes will be outlined which are based on the knowledge of conserved regions of PHA synthase structural genes and of the PHA-biosynthetic routes in bacteria as well as on the heterologous expression of these genes and on the availability of mutants impaired in the accumulation of PHA. In addition, a terminology for the designation of PHAs and of proteins and genes relevant for the metabolism of PHA is suggested.     

Endophytic Penicillium species secretes mycophenolic acid that inhibits the growth of phytopathogenic fungi

The worldwide demand for reduced and restricted use of pesticides in agriculture due to serious environmental effects, health risks and the development of pathogen resistance calls for the discovery of new bioactive compounds. In the medical field, antibiotic-resistant microorganisms have become a major threat to man, increasing mortality. Endophytes are endosymbiotic microorganisms that inhabit plant tissues without causing any visible damage to their host. Many endophytes secrete secondary metabolites with biological activity against a broad range of pathogens, making them potential candidates for novel drugs and alternative pesticides of natural origin. We isolated endophytes from wild plants in Israel, focusing on endophytes that secrete secondary metabolites with biological activity. We isolated 302 different endophytes from 30 different wild plants; 70 of them exhibited biological activity against phytopathogens. One biologically active fungal endophyte from the genus Penicillium, isolated from a squill (Urginea maritima) leaf, was further examined. Chloroform-based extraction of its growth medium was similarly active against phytopathogens. High-performance liquid chromatography separation followed by gas chromatography/mass spectrometry analysis revealed a single compound—mycophenolic acid—as the main contributor to the biological activity of the organic extract.     

Mycophenolic Acid Production by Penicillium brevicompactum on Solid Media

When grown on Czapek-Dox agar, Penicillium brevicompactum produced mycophenolic acid after a vegetative mycelium had been formed and as aerial hyphae were developing. Nutrients were still plenteous in the agar when the synthesis began. If aerial hyphal development was prevented by placing a dialysis membrane over the growing fungus, no mycophenolic acid was produced. When the dialysis membrane was peeled back and, as a consequence, production of aerial hyphae began, mycophenolic acid biosynthesis was observed. We concluded that mycophenolic acid was produced only by P. brevicompactum colonies that possessed an aerial mycelium.