cDNA isolation and functional characterization of squalene synthase gene from Ornithogalum caudatum.

As the first step of ongoing efforts to investigate the genes responsible for the biosynthesis of steroidal saponins in the medicinal plant Ornithogalum caudatum, this investigation reported the cDNA isolation, prokaryotic expression and functional characterization of squalene synthase (SQS) gene from O. caudatum for the first time. Specifically, two unigenes showing high sequence identity to SQS were retrieved from RNA-Taq data, and then a full-length OcSQS1 corresponding to the two unigenes was isolated from O. caudatum genome by a nested PCR assay. The open reading frame of OcSQS1 was 1230 bp and encoded a polypeptide of 409 aa. OcSQS1 was predicted to be a membrane-bound protein with at least four conserved motifs associated with binding, regulatory and catalytic activities of OcSQS1 and two transmembrane domains. Next, many attempts to generate soluble OcSQS1 in heterologous Escherichia coli were made, including optimization of expression conditions, application of varied expression plasmids with different tags, secretory peptides and molecular chaperones, and truncated mutation of OcSQS1. Finally, the successful availability of a soluble, truncated OcSQS1 mutant was achieved by combinational use of the utensils from the vast genetic toolbook. Moreover, this truncated OcSQS1 mutant retained the folding capability as well as its catalytic activity, converting FPP to form squalene. Importantly, the present research tentatively verified the involvement of the second transmembrane domain in the proper folding of the recombinant OcSQS1 protein.

Transcriptome-guided gene isolation and functional characterization of UDP-xylose synthase and UDP-D-apiose/UDP-D-xylose synthase families from Ornithogalum caudatum Ait.

KEY MESSAGE: The present study first identified the involvement of OcUAXS2 and OcUXS1-3 in anticancer polysaccharides biosynthesis in O. caudatum. UDP-xylose synthase (UXS) and UDP-D-apiose/UDP-D-xylose synthase (UAXS), both capable of converting UDP-D-glucuronic acid to UDP-D-xylose, are believed to transfer xylosyl residue to anticancer polysaccharides biosynthesis in Ornithogalum caudatum Ait. However, the cDNA isolation and functional characterization of genes encoding the two enzymes from O. caudatum has never been documented. Previously, the transcriptome sequencing of O. caudatum was performed in our laboratory. In this study, a total of six and two unigenes encoding UXS and UAXS were first retrieved based on RNA-Seq data. The eight putative genes were then successfully isolated from transcriptome of O. caudatum by reverse transcription polymerase chain reaction (RT-PCR). Phylogenetic analysis revealed the six putative UXS isoforms can be classified into three types, one soluble and two distinct putative membrane-bound. Moreover, the two UAXS isoenzymes were predicted to be soluble forms. Subsequently, these candidate cDNAs were characterized to be bona fide genes by functional expression in Escherichia coli individually. Although UXS and UAXS catalyzed the same reaction, their biochemical properties varied significantly. It is worth noting that a ratio switch of UDP-D-xylose/UDP-D-apiose for UAXS was established, which is assumed to be helpful for its biotechnological application. Furthermore, a series of mutants were generated to test the function of NAD+ binding motif GxxGxxG. Most importantly, the present study determined the involvement of OcUAXS2 and OcUXS1-3 in xylose-containing polysaccharides biosynthesis in O. caudatum. These data provide a comprehensive knowledge for UXS and UAXS families in plants.

cDNA cloning and expression analysis of farnesyl pyrophosphate synthase from Ornithogalum saundersiae.

Farnesyl pyrophosphate synthase (FPPS, EC 2.5.1.10) catalyzes the consecutive head-to-tail condensations of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP) to form farnesyl pyrophosphate (FPP), a key precursor of sesquiterpenoids, triterpenoids, sterols, and farnesylated proteins. Here we report the molecular cloning and functional identification of a new full-length cDNA encoding FPPS from Ornithogalum saundersiae, a potential medicinal plant that produces a promising antitumour sterol glycoside, OSW-1. An 1327 bp long unigene with an open reading frame of 1044 bp was retrieved from the transcriptome sequencing of O. saundersiae. The full-length FPPS cDNA, designated OsaFPPS, was isolated from O. saundersiae with gene-specific primers. The resultant OsaFPPS encodes a 347-amino acids protein with a calculated molecular mass of 40,085.6 Da, and a theoretical isoelectric point of 5.01. Phylogenetic tree analysis indicated that OsaFPPS belongs to the plant FPPS super-family. Expression of soluble OsaFPPS in E. coli was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Functional analysis of the purified OsaFPPS protein was carried out using IPP and DMAPP as substrates, and the product was unambiguously determined by gas chromatography-mass spectrometry (GC-MS) analyses.