The biosynthesis of these fungal polyketides involves a ..
Previous study have succeeded in heterologous production of fungal polyketides by involving A. nidulans PPtase, revealing that PPtase worked well on ACP of 6-MSAS and LNKS origining from P. patulum and A. terrus, respectively [,]. In this study, A. nidulans PPtase reacting with ACP of M. purpureus was also proven. These results indicated that A. nidulans PPtase probably work on ACPs from a wide range of filamentous fungi.
Functional analysis of fungal polyketide biosynthesis genes.
Polyketides are one of the most important classes of secondary metabolites and usually make good drugs. Currently, heterologous production of fungal polyketides for developing a high potential industrial application system with high production capacity and pharmacutical feasibility was still at its infancy. Pichia pastoris is a highly successful system for the high production of a variety of heterologous proteins. In this work, we aim to develop a P. pastoris based in vivo fungal polyketide production system for first time and evaluate its feasibility for future industrial application.
Three strains, GS115, GS115-ATX and GS115-NpgA-ATX, were cultivated under same conditions. After methanol induced expression for 36 h, the products were extracted and analyzed by HPLC analysis. As shown in Figure , GS115-NpgA-ATX produced a specfic compound emerged as a sharp peak in HPLC chromatogram at 16.7 min compared with GS115 and GS115-ATX, which had the same retention time as an authentic sample of 6-MSA under this elution condition. To further confirm the structure of the compound, the extracts were purified by TLC (petroleum ether:ethyl acetate=3:1 (v/v), 1% acetic acid) and extracted and freeze-dried for further EI-MS and NMR analysis. The EI-MS analysis was performed on an Agilent G2577A mass spectrometer, establishing the molecular formulae as C8H8O3 for 6-MSA (with the M+ ion at m/z=152.0474, 152.0473 calculated) absolutely accorded with the standard 6-MSA (Figure A). The freeze-dried sample dissolved in deuterated DMSO for 1HNMR analysis. The results that 1HNMR (400 MHz, DMSO-d6),δH=6.37 (1H, m, H-3), 6.94 (1H, m, H-4), 6.46 (1H, m, H-5), 2.52 (3H, s, CH3-6) conformed with 1HNMR spectrum of standard 6-MSA and further confirmed the compound (Figure B). For all three tested strains, only GS115-NpgA-ATX produced 6-MSA but not GS115-ATX and wild type GS115, proving that 6-MSAS could be well modified by PPtase and it only works for 6-MSA biosynthesis after PPtase modification. Furthermore, transcription analysis of GS115 and GS115-NpgA-ATX were carried out after 24 hours induction. Three pairs of primers, 5AOX1/3AOX1, NpgAF/NpgAR1, BstpF/AtxR, were used to test gene transcriptions of AOX1, npgA and atX. The wild type GS115 only generated the correct nucleic acid size of 2.2 kb for the AOX1, while GS115-NpgA-ATX formed other two desired PCR products (1.3 kb for npgA and 1.9 kb for atX) besides the AOX1 (Figure C), indicating that both npgA and atX transcripted effectively in the recombinant strain GS115-NpgA-ATX.
Fungal polyketides are of intense ..
N2 - Resorcylic acid lactones and dihydroxyphenylacetic acid lactones represent important pharmacophores with heat shock response and immune system modulatory activities. The biosynthesis of these fungal polyketides involves a pair of collaborating iterative polyketide synthases (iPKSs): a highly reducing iPKS with product that is further elaborated by a nonreducing iPKS (nrPKS) to yield a 1,3- benzenediol moiety bridged by a macrolactone. Biosynthesis of unreduced polyketides requires the sequestration and programmed cyclization of highly reactive poly-β-ketoacyl intermediates to channel these uncommitted, pluripotent substrates to defined subsets of the polyketide structural space. Catalyzed by product template (PT) domains of the fungal nrPKSs and discrete aromatase/cyclase enzymes in bacteria, regiospecific first-ring aldol cyclizations result in characteristically different polyketide folding modes. However, a few fungal polyketides, including the dihydroxyphenylacetic acid lactone dehydrocurvularin, derive from a folding event that is analogous to the bacterial folding mode. The structural basis of such a drastic difference in the way a PT domain acts has not been investigated until now. We report here that the fungal vs. bacterial folding mode difference is portable on creating hybrid enzymes, and we structurally characterize the resulting unnatural products. Using structure-guided active site engineering, we unravel structural contributions to regiospecific aldol condensations and show that reshaping the cyclization chamber of a PT domain by only three selected point mutations is sufficient to reprogram the dehydrocurvularin nrPKS to produce polyketides with a fungal fold. Such rational control of first-ring cyclizations will facilitate efforts to the engineered biosynthesis of novel chemical diversity from natural unreduced polyketides.
Polyketide Biosynthesis - University of Bristol
N2 - Sphinganine-analog mycotoxins (SAMT) are polyketide-derived natural products produced by a number of plant pathogenic fungi and are among the most economically important mycotoxins. The toxins are structurally similar to sphinganine, a key intermediate in the biosynthesis of ceramides and sphingolipids, and competitive inhibitors for ceramide synthase. The inhibition of ceramide and sphingolipid biosynthesis is associated with several fatal diseases in domestic animals and esophageal cancer and neural tube defects in humans. SAMT contains a highly reduced, acyclic polyketide carbon backbone, which is assembled by a single module polyketide synthase. The biosynthesis of SAMT involves a unique polyketide chain-releasing mechanism, in which a pyridoxal 5′-phosphate-dependent enzyme catalyzes the termination, offloading and elongation of the polyketide chain. This leads to the introduction of a new carbon-carbon bond and an amino group to the polyketide chain. The mechanism is fundamentally different from the thioesterase/cyclase- catalyzed polyketide chain releasing found in bacterial and other fungal polyketide biosynthesis. Genetic data suggest that the ketosynthase domain of the polyketide synthase and the chain-releasing enzyme are important for controlling the final product structure. In addition, several post-polyketide modifications have to take place before SAMT become mature toxins.