Characterization of γ-Cadinene Enzymes in Ganoderma lucidum and Ganoderma sinensis from Basidiomycetes Provides Insight into the Identification of Terpenoid Synthases

Abstract

Enzymes boost protein engineering, directed evolution, and the biochemical industry and are also the cornerstone of metabolic engineering. Basidiomycetes are known to produce a large variety of terpenoids with unique structures. However, basidiomycetous terpene synthases remain largely untapped. Therefore, we provide a modeling method to obtain specific terpene synthases. Aided by bioinformatics analysis, three γ-cadinene enzymes from Ganoderma lucidum and Ganoderma sinensis were accurately predicted and identified experimentally. Based on the highly conserved amino motifs of the characterized γ-cadinene enzymes, the enzyme was reassembled as model 1. Using this model as a template, 67 homologous sequences of the γ-cadinene enzyme were screened from the National Center for Biotechnology Information (NCBI). According to the 67 sequences, the same gene structure, and similar conserved motifs to model 1, the γ-cadinene enzyme model was further improved by the same construction method and renamed as model 2. The results of bioinformatics analysis show that the conservative regions of models 1 and 2 are highly similar. In addition, five of these sequences were verified, 100% of which were γ-cadinene enzymes. The accuracy of the prediction ability of the γ-cadinene enzyme model was proven. In the same way, we also reanalyzed the identified Δ⁶-protoilludene enzymes in fungi and (-)-α-bisabolol enzymes in plants, all of which have their own unique conserved motifs. Our research method is expected to be used to study other terpenoid synthases with a similar or the same function in basidiomycetes, ascomycetes, bacteria, and plants and to provide rich enzyme resources.

Figure 1

Gene structure-phylogenetic tree and sequence alignment of γ-CSs. (A) Gene structure-phylogenetic tree of γ-CS sequences and nine-exon genes. (B) Amino acid sequence alignments of six γ-CSs. Amino acids range from low (blue) to high (red) conservation.

Figure 2

Characterization of γ-CSs from G. lucidum and G. sinensis in E. coli. (A) Expression of various γ-CSs from G. lucidum and G. sinensis. Lane M, molecular weight marker protein; S, soluble fraction of total cell extracts; and I, insoluble fraction of total cell extracts. (B) GC–MS chromatograms of cultural supernatants of strains GlSTS6, GsSTS43, and GsSTS45b and the control strains pET32a _ctrl and the positive STC9_posi. (C) Prediction and cloning of a gene in the G. sinensis STS family. The predicted gene was GsSTS45. The cloned and reannotated genes were GsSTS45a and GsSTS45b, and only GsSTS45b revealed to be functional.

Figure 3

Homology model 2 of γ-CS was constructed. Model 2 based on conservative regions of 67 sequences (see the Supporting Information) retrieved by model 1 (see the Supporting Information) in NCBI. Red represented 100% conservatism, and blue represents >80% conservatism of amino acid.

Figure 4

Verification of predictive ability for homology model 2, and γ-CSs were selected according to a principle that the amino acid sequence in each branch of the phylogenetic tree is most similar to the red and blue amino acids of model 2. (A) Expression of five selected γ-CSs. Lane M, molecular weight marker protein; S, soluble fraction of total cell extracts; and I, insoluble fraction of total cell extracts. (B) GC–MS chromatograms of cultural supernatants of five γ-CS strains. (C) Production of γ-cadinene for five γ-CSs. STC9 production of γ-cadinene is set to 100%; error bars indicate standard deviations determined from triplicates.

Figure 5

Gene structure-phylogenetic tree and homology model of Δ⁶-protoilludene synthases. (A) Gene structure-phylogenetic tree of 15 characterized Δ⁶-protoilludene synthases (see the Supporting Information). (B) Homology model of Δ⁶-protoilludene synthases. The modeling method is the same as the γ-CS; red represents 100% conservatism, and blue represents >80% conservatism of the amino acid.

Figure 6

Sequence alignment and homology model of BOSs. (A) Amino acid sequence alignments of four BOSs. MrBOS, Matricaria recutita (AIG92846.1); AaBOS, Artemisia annua (AFV40969.1); EeBOS, E. erythropappus (DC) McLeisch (AYJ71561.1); and CcBOS, C. cardunculus var. scolymus (XP_024994640.1). Amino acids range from low (blue) to high (red) conservation. (B) Homology model of BOSs. The modeling method is the same as the γ-CS, red represents 100% conservatism.