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Background <p>Diterpenes derived from geranylgeranyl diphosphate include the pimarane family, a class of tricyclic diterpenoids widely distributed in plants and fungi. Previous studies on <i>Aspergillus nidulans</i> identified a biosynthetic gene cluster containing a diterpene cyclase that produces <i>ent</i>-pimara-8(14),15-diene, whereas subsequent biochemical analysis suggested the enzyme instead generates an isopimarane scaffold.</p> Results <p>In this study, we have reexplored this diterpene biosynthetic pathway by using a multi-cluster deletion strain to reduce the metabolite background and a modified version of the highly inducible <i>alcA</i> promoter system to drive expression of a transcription factor in the cluster that includes the diterpene cyclase gene. The transcription factor, in turn, drives expression of all the genes of the cluster. Induction of expression resulted in production of compounds not seen with prior methods. Liquid chromatography-mass spectrometry (LC-MS) and Nuclear magnetic resonance (NMR) analysis has enabled us to identify dipleosmarane (<b>3</b>), a new dimeric isopimarane derivative, together with six known isopimarane diterpenoids (<b>1</b>, <b>2</b>, <b>4a</b>, <b>4b</b>, <b>5</b>, <b>6</b>). Gene deletion experiments targeting the terpene cyclase (<i>pbcA</i>) and geranylgeranyl pyrophosphate synthase (<i>pbcB</i>) confirmed that these metabolites originate from this cluster.</p> Conclusions <p>Our findings support the conclusion that the <i>A. nidulans</i> diterpene cyclase primarily synthesizes an isopimarane scaffold rather than an <i>ent</i>-pimarane derivative. Furthermore, the combination of transcription factor activation and a multi-cluster deletion background proved effective for revealing previously undetected metabolites from this pathway. These findings provide new insight into fungal diterpenoid biosynthesis and establish a useful platform for further genetic dissection and engineering of isopimarane biosynthetic pathways in <i>Aspergillus</i>.</p> Graphical Abstract <p></p>

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Discovery of a dimeric isopimarane derivative by reexploring a labdane-related diterpenoid biosynthetic gene cluster in Aspergillus nidulans

  • 期刊论文

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  • 发表日期：

    2026年7月13日

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  • Shu-Yi Lin,
  • Cory B. Jenkinson,
  • Quoc-Dung Tran Huynh,
  • C. Elizabeth Oakley,
  • Ching-Kuo Lee,
  • Haiyao Ma,
  • Berl R. Oakley,
  • Clay C. C. Wang

摘要

Background

Diterpenes derived from geranylgeranyl diphosphate include the pimarane family, a class of tricyclic diterpenoids widely distributed in plants and fungi. Previous studies on Aspergillus nidulans identified a biosynthetic gene cluster containing a diterpene cyclase that produces ent-pimara-8(14),15-diene, whereas subsequent biochemical analysis suggested the enzyme instead generates an isopimarane scaffold.

Results

In this study, we have reexplored this diterpene biosynthetic pathway by using a multi-cluster deletion strain to reduce the metabolite background and a modified version of the highly inducible alcA promoter system to drive expression of a transcription factor in the cluster that includes the diterpene cyclase gene. The transcription factor, in turn, drives expression of all the genes of the cluster. Induction of expression resulted in production of compounds not seen with prior methods. Liquid chromatography-mass spectrometry (LC-MS) and Nuclear magnetic resonance (NMR) analysis has enabled us to identify dipleosmarane (3), a new dimeric isopimarane derivative, together with six known isopimarane diterpenoids (1, 2, 4a, 4b, 5, 6). Gene deletion experiments targeting the terpene cyclase (pbcA) and geranylgeranyl pyrophosphate synthase (pbcB) confirmed that these metabolites originate from this cluster.

Conclusions

Our findings support the conclusion that the A. nidulans diterpene cyclase primarily synthesizes an isopimarane scaffold rather than an ent-pimarane derivative. Furthermore, the combination of transcription factor activation and a multi-cluster deletion background proved effective for revealing previously undetected metabolites from this pathway. These findings provide new insight into fungal diterpenoid biosynthesis and establish a useful platform for further genetic dissection and engineering of isopimarane biosynthetic pathways in Aspergillus.

Graphical Abstract