Cloning and characterization of squalene synthase gene from Fusarium fujikuroi (Saw.) Wr.

Abstract

The gene encoding squalene synthase (GfSQS) was cloned from Fusarium fujikuroi (Gibberella fujikuroi MP-C) and characterized. The cloned genomic DNA is 3,267 bp in length, including the 5'-untranslated region (UTR), 3'-UTR, four exons, and three introns. A noncanonical splice-site (CA-GG, or GC-AG) was found at the first intron. The open reading frame of the gene is 1,389 bp in length, corresponding to a predicted polypeptide of 462 amino acid residues with a MW 53.4 kDa. The predicted GfSQS shares at least four conserved regions involved in the enzymatic activity with the SQSs of varied species. The recombinant protein was expressed in E. coli and detected by SDS-PAGE and western blot. GC-MS analysis showed that the wild-type GfSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene, while the mutant mGfSQS (D82G) lost total activity, supporting the prediction that the aspartate-rich motif (DTXED) in the region I of SQS is essential for binding of the diphosphate substrate.

Heat Stress Modulates Mycelium Growth, Heat Shock Protein Expression, Ganoderic Acid Biosynthesis, and Hyphal Branching of Ganoderma lucidum via Cytosolic Ca 2+

“…Previous studies have focused on the isolation and structural determination of GA and have attempted to understand the mechanisms underlying their action (4), but less work has been conducted to evaluate their biosynthesis. Several researchers have demonstrated the key biosynthetic enzyme genes in GA biosynthesis (7,8). Some researchers have also reported that adding inducers, such as acetic acid and methyl jasmonate, in medium can increase GA accumulation (9, 10).…”

Cloning and Characterization of the Glycoside Hydrolases That Remove Xylosyl Groups from 7-β-xylosyl-10-deacetyltaxol and Its Analogues

“…The genomic DNA was extracted via the genomic DNA isolation mini-prep method (37), and the total RNA was isolated using the RNeasy plant mini kit (Qiagen, Shanghai, China).…”

Directed optimization of a newly identified squalene synthase from Mortierella alpine based on sequence truncation and site-directed mutagenesis

“…As a key precursor of isoprenoids, SQ is synthesized from FPP by SQS (Fig. 1), which has been identified in human beings [36,38], animals [14,24,34], plants [11,12,18,26,42,50], fungi [16,20,51,52], bacteria [21]. SQS is commonly considered to be a crucial enzyme and a potential regulatory branch node that controls carbon flux towards the triterpenes and sterols biosynthesis.…”

“…Besides, although amino acid residues in the C-terminal region show a low level of sequence identity among all SQS proteins (Fig. 2), this region is very hydrophobic among all SQS enzymes and thus functions as an anchor in the putative endoplasmic reticulum membrane [53], which was also predicted by TMHMM and SPLIT 4.0 bioinformatics programs. Furthermore, the phylogenetic tree suggested that the fungal enzymes were clustered into one group (Fig.…”