Unravelling fungal biosynthetic potential by heterologous expression of silent gene clusters in Aspergillus nidulans and LC-MS-based metabolomic analysis

• Applicant: Professor Dr. Shu-Ming Li
• Subject Area: Pharmacy
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 410588509

• Final Report Year: 2023

Final Report Abstract

In this project we combined the power of genomics and synthetic biology with an integrated metabolomic profiling tool box to express silent/cryptic genes and to elucidate secondary metabolite (SM) pathways. Putative biosynthetic gene clusters (BGCs) from Penicillium crustosum, Pestalotiopsis fici, Aspergillus ruber, Penicillium palitans, Penicillium roqueforti and Trichoderma applanatum were first cloned in E. coli-Saccharomyces-Aspergillus shuttle vectors by PCR-based homologues recombination in E. coli or yeast and the constructs were then successfully expressed in Aspergillus nidulans LO8030. Expression of such clusters led to accumulation of pathway products, which were identified by isolation and structural elucidation. Subsequent gene deletion and feeding experiments as well as biochemical characterization of the recombinant key enzymes have been used for elucidation of the reaction steps in the 15 biosynthetic pathways, which revealed the presence of novel enzymes or reactions in their biosynthesis. These include the formation mechanism of peniphenones and penilactones in P. crustosum, the first fungal enzyme FnsA for the flavonoid biosynthesis in P. fici, the bifunctional PKS OesA from P. crustosum, the lactone-forming bridge enzyme-like protein AnuG from P. roqueforti and the P450 enzyme VdoD from P. palitans for meta-hydroxylation at a monosubstituted benzene ring.

Publications

• Formation of Terrestric Acid in Penicillium crustosum Requires Redox-Assisted Decarboxylation and Stereoisomerization. Organic Letters, 22(1), 88-92.
  Fan, Jie; Liao, Ge; Ludwig-Radtke, Lena; Yin, Wen-Bing & Li, Shu-Ming
  
• Genomic Locus of a Penicillium crustosum Pigment as an Integration Site for Secondary Metabolite Gene Expression. ACS Chemical Biology, 14(6), 1227-1234.
  Kindinger, Florian; Nies, Jonas; Becker, Anke; Zhu, Tianjiao & Li, Shu-Ming
  
• Peniphenone and Penilactone Formation in Penicillium crustosum via 1,4-Michael Additions of ortho-Quinone Methide from Hydroxyclavatol to γ-Butyrolactones from Crustosic Acid. Journal of the American Chemical Society, 141(10), 4225-4229.
  Fan, Jie; Liao, Ge; Kindinger, Florian; Ludwig-Radtke, Lena; Yin, Wen-Bing & Li, Shu-Ming
  
• Biosynthesis of the Prenylated Salicylaldehyde Flavoglaucin Requires Temporary Reduction to Salicyl Alcohol for Decoration before Reoxidation to the Final Product. Organic Letters, 22(6), 2256-2260.
  Nies, Jonas; Ran, Huomiao; Wohlgemuth, Viola; Yin, Wen-Bing & Li, Shu-Ming
  
• Isocoumarin formation by heterologous gene expression and modification by host enzymes. Organic & Biomolecular Chemistry, 18(26), 4946-4948.
  Xiang, Pan; Ludwig-Radtke, Lena; Yin, Wen-Bing & Li, Shu-Ming
  
• Prenylation and Dehydrogenation of a C2-Reversely Prenylated Diketopiperazine as a Branching Point in the Biosynthesis of Echinulin Family Alkaloids in Aspergillus ruber. ACS Chemical Biology, 16(1), 185-192.
  Nies, Jonas & Li, Shu-Ming
  
• Biosynthesis of Viridicatol in Penicillium palitans Implies a Cytochrome P450-Mediated meta Hydroxylation at a Monoalkylated Benzene Ring. Organic Letters, 24(1), 262-267.
  Zhang, Zheng-Xi; Li, Zhang-Hai; Yin, Wen-Bing & Li, Shu-Ming
  
• Conversion of viridicatic acid to crustosic acid by cytochrome P450 enzyme-catalysed hydroxylation and spontaneous cyclisation. Applied Microbiology and Biotechnology, 105(24), 9181-9189.
  Zhou, Jenny & Li, Shu-Ming
  
• Formation of 3-Orsellinoxypropanoic Acid in Penicillum crustosum is Catalyzed by a Bifunctional Nonreducing Polyketide Synthase. Organic Letters, 24(1), 462-466.
  Xiang, Pan & Li, Shu-Ming
  
• Fungal benzene carbaldehydes: occurrence, structural diversity, activities and biosynthesis. Natural Product Reports, 38(1), 240-263.
  Ran, Huomiao & Li, Shu-Ming
  
• Heterologous expression of a single fungal HR-PKS leads to the formation of diverse 2-alkenyl-tetrahydropyrans in model fungi. Organic & Biomolecular Chemistry, 19(38), 8377-8383.
  Lyu, Hai-Ning; Zhang, Jinyu; Zhou, Shuang; Liu, Hong-Wei; Zhuang, Wen-Ying; Li, Shu-Ming & Yin, Wen-Bing
  
• Precursor Supply Increases the Accumulation of 4-Hydroxy-6-(4-hydroxyphenyl)-α-pyrone after NRPS–PKS Gene Expression. Journal of Natural Products, 84(8), 2380-2384.
  Li, Wen; Fan, Jie; Liao, Ge; Yin, Wen-Bing & Li, Shu-Ming
  
• A fungal NRPS-PKS enzyme catalyses the formation of the flavonoid naringenin. Nature Communications, 13(1).
  Zhang, Hongjiao; Li, Zixin; Zhou, Shuang; Li, Shu-Ming; Ran, Huomiao; Song, Zili; Yu, Tao & Yin, Wen-Bing
  
• Biosynthesis of Annullatin D in Penicillium roqueforti Implies Oxidative Lactonization between Two Hydroxyl Groups Catalyzed by a BBE-like Enzyme. Organic Letters, 24(32), 6072-6077.
  Xiang, Pan; Kemmerich, Bastian; Yang, Li & Li, Shu-Ming
  
• Biosynthesis of Xylariolide D in Penicillium crustosum Implies a Chain Branching Reaction Catalyzed by a Highly Reducing Polyketide Synthase. Journal of Fungi, 8(5), 493.
  Stierle, Sina A. & Li, Shu-Ming
  
• Pretrichodermamide A Biosynthesis Reveals the Hidden Diversity of Epidithiodiketopiperazines. Angewandte Chemie, 135(18).
  Fan, Jie; Ran, Huomiao; Wei, Peng‐Lin; Li, Yuanyuan; Liu, Huan; Li, Shu‐Ming; Hu, Youcai & Yin, Wen‐Bing