Functional heterologous expression of the reversible Cu-decarboxylase from the lichen, Cladonia uncialis

Abstract

Despite the isolation of over 1000 known bioactive lichen mycobiont-derived secondary metabolites (SMs), understanding the genetic basis of their biosynthesis remains elusive. Biosynthetic gene clusters (BGCs) have been tentatively linked to chemical structures, with core genes such as polyketide synthases (PKSs) surrounded by accessory genes like decarboxylases. In this study, we focused on a decarboxylase gene from the genome of the lichen cladonia uncialis (named as Cu-decarboxylase) to elucidate its role in SM biosynthesis. A 963 bp gene was cloned from C. uncialis and expressed in Escherichia coli (BL21(DE3) cells using the pQE80L expression vector. The resulting 35 kDa protein was purified by applying a Ni⁺-NTA column using an FPLC system. Functional activity assays revealed the decarboxylation and reversible carboxylation of resorcinol to 2,4-dihydroxybenzoic acid and orcinol to orsellinic acid. This suggests a potential role for this Cu-decarboxylase in SM biosynthesis.

Furthermore, the lack of activity on substrates like anthranilic acid and aniline highlighted the importance of the phenolic OH group in facilitating these reactions. The 3D protein structure was predicted with AlphaFold3, based on sequence similarity with a known decarboxylases and revealed the importance of a zinc cofactor for the catalytic activity of the enzyme. The optimization of the reaction conditions, particularly for orsellinic acid production from orcinol, may enhance conversion rates and offer a viable route for industrial-scale production of bioactive compounds. This study marks the first known instance of functional heterologous expression of a non-codon-optimized gene isolated from lichen in E. coli.

Keywords

Isolation / Secondary / Bioactive compounds / Polyketides / Carboxylation / Decarboxylation

Data Availability Statement

All data presented in this manuscript, including plasmids and gene sequences, is available free of charge by contacting the corresponding author.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT Authorship Contribution Statement

Harman Gill: Writing - original draft, Investigation, Formal analysis. John L. Sorensen: Writing - review & editing, Supervision, Conceptualization.

Acknowledgments

We would like to acknowledge Dr. Hamidreza Karbalaeiheidari (Department of Chemistry, U of Manitoba), for his guidance in cloning, and Dr. Nediljko Budisa (Department of Chemistry, U of Manitoba) for providing the expression plasmid and granting access to the FPLC instrument in their lab.

Financial support from an NSERC Discovery Grant (RGPIN-2017-04958) to JLS for this research is gratefully acknowledged. Funding to HG from the U of Manitoba Faculty of Science Enhancement of Graduate Stipends Program is acknowledged.

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