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UCLA UCLA Electronic Theses and Dissertations UCLA UCLA Electronic Theses and Dissertations Title Bridging the Gap: Studying Sequence to Product Correlation among Fungal Polyketide Synthases Permalink https://escholarship.org/uc/item/8g12b08v Author Zabala, Angelica Obusan Publication Date 2014 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA Los Angeles Bridging the Gap: Studying Sequence to Product Correlation among Fungal Polyketide Synthases A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Chemical Engineering by Angelica Obusan Zabala 2014 © Copyright by Angelica Obusan Zabala 2014 ii ABSTRACT OF THE DISSERTATION Bridging the Gap: Studying Sequence to Product Correlation among Fungal Polyketide Synthases by Angelica Obusan Zabala Doctor of Philosophy in Chemical Engineering University of California, Los Angeles, 2014 Professor Yi Tang, Chair Polyketide synthases (PKSs) catalyze the production of important pharmaceuticals, including statins and antibiotics. Fungi are some of the most prolific producers of these compounds but still have untapped potential as demonstrated by the abundant secondary metabolic clusters uncovered from the genome mining efforts. In order to better understand the correlation between the fungal Type I PKSs and their products for predictive and engineering purposes, it is necessary to biochemically characterize these enzyme systems. To do so, two distinct approaches were used. In the first bottom-up approach, we demonstrated that we can take the sequence of a cluster and elucidate its products by selective activation. This led to the discovery of new compounds iii belonging to the azaphilone group of natural products. We studied key aspects on the biosynthesis of this natural product group, particularly the formation of the characteristic pyrano-quinone core. This helped facilitate the discovery of new azaphilone clusters from the broad fungal genome space. In the second top-down approach, we studied the biosynthesis of the potent protein-transport inhibitor Brefeldin A. By sequencing the producing organism and using detailed bioinformatic analysis, we localized the cluster responsible for its production. More importantly, we provide new insights in the programming of these enigmatic HRPKSs, particularly in the permutative use of the reductive domains and the chain-length control by an in trans thiohydrolase. iv The dissertation of Angelica Obusan Zabala is approved. James Liao Neil Garg Yi Tang, Committee Chair University of California, Los Angeles 2014 v Table of Contents 1 Introduction ............................................................................................................................. 1 1.1 Natural products: Spotlight on Polyketides ...................................................................... 1 1.2 Polyketide synthases ........................................................................................................ 3 1.2.1 Fungal Iterative Polyketide Synthases ...................................................................... 7 1.3 Methods for studying PKS gene clusters in the genomic era ......................................... 15 1.3.1 Genetic manipulation of native host ....................................................................... 15 1.3.2 Heterologous expression of clusters and individual genes ..................................... 17 1.3.3 In vitro reconstitution of protein activity ................................................................ 18 1.4 Engineering PKSs and tailoring enzymes for NP diversification .................................. 19 1.4.1 Engineering PKSs ................................................................................................... 19 1.4.2 Engineering tailoring enzymes ............................................................................... 28 1.5 Motivation for study ....................................................................................................... 33 2 Results ................................................................................................................................... 34 2.1 Discovery and Biosynthesis of Azanigerones from Aspergillus niger ATCC 1015 ...... 34 2.1.1 Genome mining in Aspergillus niger ATCC 1015 ................................................. 34 2.1.2 Characterization of a Silent Azaphilone Gene Cluster from Aspergillus niger ATCC 1015 Reveals a Hydroxylation-Mediated Pyran-Ring Formation.” .......................... 42 2.1.3 Heterologous expression of aza proteins ................................................................ 68 2.1.4 Bioactivity assays on azanigerone A ...................................................................... 73 vi 2.2 Biosynthesis of Brefeldin ............................................................................................... 74 2.2.1 Significance and activity of Brefeldin A ................................................................ 74 2.2.2 Genome mining for BFA-producing cluster in Eupenicillium brefeldianum ATCC 58665 75 2.2.3 Fungal Polyketide Synthase Product Chain-Length Control by Partnering Thiohydrolase ........................................................................................................................ 82 3 Conclusion ........................................................................................................................... 119 4 Appendix ............................................................................................................................. 122 4.1 Supplementary information for Section 2.1.2 .............................................................. 122 4.2 Supplementary information for Section 2.1.3. ............................................................. 158 4.3 Supplementary information for Section 2.2.3. ............................................................. 162 5 References ........................................................................................................................... 187 vii List of Figures Figure 1.1. Polyketide natural products from different sources. ..................................................... 2 Figure 1.2. Divergence between PKS and FAS functions. ............................................................. 4 Figure 1.3. Schematic representation of the biosynthetic mechanism of different PKSs ............... 6 Figure 1.4. Biosynthetic scheme for the activity of IPKSs. ............................................................ 7 Figure 1.5. Representative compounds produced by HRPKSs....................................................... 8 Figure 1.6. Biosynthesis of lovastatin. .......................................................................................... 10 Figure 1.7. Representative compounds produced by NRPKSs..................................................... 11 Figure 1.8. Representative compounds produced by tandem HRPKS-NRPKS systems. ............ 13 Figure 1.9. Biosynthesis of the hypothemycin precursor DHZ .................................................... 15 Figure 1.10. Increasing Type III PKS product chain length ......................................................... 23 Figure 1.11. Protein engineering of a “universal” glycosyltransferase. ....................................... 30 Figure 2.1. Secondary metabolites produced by A. niger. ............................................................ 36 Figure 2.2. Breakdown of PKSs found from genome mining of A. niger ATCC 1015. .............. 37 Figure 2.3. Examples of azaphilone compounds isolated from fungi........................................... 44 Figure 2.4. Comparison of the A. niger aza cluster with the A. nidulans afo cluster and their respective products........................................................................................................................ 46 Figure 2.5. Transcriptional analysis by RT-PCR of the aza genes in A. niger WT and the activated T1 strain ......................................................................................................................... 48 Figure 2.6. Metabolic profiles of the activated A. niger T1 strain on days 2, 4 and 7 and of the WT on day 2.................................................................................................................................. 49 Figure 2.7. Structures of 2 - 7 isolated from the activated A. niger T1 strain. ............................. 50 Figure 2.8. Knockout of azaB demonstrates convergent mode of PKS collaboration. ................ 54 viii Figure 2.9. In vitro reaction with AzaH. ....................................................................................... 55 Figure 2.10. Proposed biosynthetic pathway for production of azanigerones from the aza cluster. ....................................................................................................................................................... 58 Figure 2.11. Cloning strategy to construct the azaA-harboring plasmid, pAZ103. ...................... 69 Figure 2.12. HPLC analysis of Day 3 cultures of S.c. with AzaA and the control strain. Compound 6 is exclusively produced by the AzaA-expressing strain. ........................................ 70 Figure 2.13. Conversion of 8 to 9 ................................................................................................. 71 Figure 2.14. AzaE controls spontaneous cyclization of AzaA product. ......................................
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