RESUMO
Farnesyl pyrophosphate synthase (FPPS) catalyzes the synthesis of C15 farnesyl diphosphate (FPP) from C5 dimethylallyl diphosphate (DMAPP) and two or three C5 isopentenyl diphosphates (IPPs). FPP is an important precursor for the synthesis of isoprenoids and is involved in multiple metabolic pathways. Here, farnesyl pyrophosphate synthase from Sporobolomyces pararoseus NGR (SpFPPS) was isolated and expressed by the prokaryotic expression system. The SpFPPS full-length genomic DNA and cDNA are 1566 bp and 1053 bp, respectively. This gene encodes a 350-amino acid protein with a predicted molecular mass of 40.33 kDa and a molecular weight of 58.03 kDa (40.33 kDa + 17.7 kDa), as detected by SDS-PAGE. The function of SpFPPS was identified by induction, purification, protein concentration and in vitro enzymatic activity experiments. Structural analysis showed that Y90 was essential for chain termination and changing the substrate scope. Site-directed mutation of Y90 to the smaller side-chain amino acids alanine (A) and lysine (K) showed in vitro that wt-SpFPPS catalyzed the condensation of the substrate DMAPP or geranyl diphosphate (GPP) with IPP at apparent saturation to synthesize FPP as the sole product and that the mutant protein SpFPPS-Y90A synthesized FPP and C20 geranylgeranyl diphosphate (GGPP), while SpFPPS-Y90K hydrolyzed the substrate GGPP. Our results showed that FPPS in S. pararoseus encodes the SpFPPS protein and that the amino acid substitution at Y90 changed the distribution of SpFPPS-catalyzed products. This provides a baseline for potentially regulating SpFPPS downstream products and improving the carotenoid biosynthesis pathway.
RESUMO
Salinity stress is one of the most serious environmental issues in agricultural regions worldwide. Excess salinity inhibits root growth of various crops, and results in reductions of yield. It is of crucial to understand the molecular mechanisms mediating salinity stress responses for enhancing crops' salt tolerance. Marine red yeast Sporobolomyces pararoseus should have evolved some unique salt-tolerant mechanism, because they long-term live in high-salt ecosystems. However, little research has conducted so far by considering S. pararoseus as model microorganisms to study salt-tolerant mechanisms. Here, we successfully integrated metabolomics with transcriptomic profiles of S. pararoseus in response to salinity stress. Screening of metabolite features with untargeted metabolic profiling, we characterized 4862 compounds from the LC-MS/MS-based datasets. The integrated results showed that amino acid metabolism, carbohydrate metabolism, and lipid metabolism is significantly enriched in response to salt stress. Co-expression network analysis showed that 28 genes and 8 metabolites play an important role in the response of S. pararoseus, which provides valuable clues for subsequent validation. Together, the results provide valuable information for assessing the central metabolism of mediating salt responses in S. pararoseus and offer inventories of target genes for salt tolerance improvement via genetic engineering.
Assuntos
Basidiomycota/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Basidiomycota/genética , Basidiomycota/metabolismo , Metabolismo dos Carboidratos , Cromatografia Líquida , Estresse Salino/genética , Cloreto de Sódio/toxicidade , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Sporobolomyces pararoseus is regarded as an oleaginous red yeast, which synthesizes numerous valuable compounds with wide industrial usages. This species hold biotechnological interests in biodiesel, food and cosmetics industries. Moreover, the ballistospores-shooting promotes the colonizing of S. pararoseus in most terrestrial and marine ecosystems. However, very little is known about the basic genomic features of S. pararoseus. To assess the biotechnological potential and ballistospores-shooting mechanism of S. pararoseus on genome-scale, the whole genome sequencing was performed by next-generation sequencing technology. RESULTS: Here, we used Illumina Hiseq platform to firstly assemble S. pararoseus genome into 20.9 Mb containing 54 scaffolds and 5963 predicted genes with a N50 length of 2,038,020 bp and GC content of 47.59%. Genome completeness (BUSCO alignment: 95.4%) and RNA-seq analysis (expressed genes: 98.68%) indicated the high-quality features of the current genome. Through the annotation information of the genome, we screened many key genes involved in carotenoids, lipids, carbohydrate metabolism and signal transduction pathways. A phylogenetic assessment suggested that the evolutionary trajectory of the order Sporidiobolales species was evolved from genus Sporobolomyces to Rhodotorula through the mediator Rhodosporidiobolus. Compared to the lacking ballistospores Rhodotorula toruloides and Saccharomyces cerevisiae, we found genes enriched for spore germination and sugar metabolism. These genes might be responsible for the ballistospores-shooting in S. pararoseus NGR. CONCLUSION: These results greatly advance our understanding of S. pararoseus NGR in biotechnological potential and ballistospores-shooting, which help further research of genetic manipulation, metabolic engineering as well as its evolutionary direction.
Assuntos
Basidiomycota/genética , Genoma Fúngico , Filogenia , Sequenciamento Completo do Genoma , Basidiomycota/metabolismo , Biotecnologia , Metabolismo dos Carboidratos , Carotenoides/metabolismo , Genômica , Metabolismo dos Lipídeos , Análise de Sequência de RNARESUMO
Carotenoids, a group of natural pigments, have strong antioxidant properties and act as precursors to vitamin A, which have garnered attention from industry and researchers. Sporobolomyces pararoseus represents a hyper-producer of carotenoids, mainly including ß-carotene, torulene, and torularhodin. Geranylgeranyl diphosphate synthase (GGPPS) is regarded as a key enzyme in the carotenoid biosynthesis pathway. However, the precise nature of the gene encoding GGPPS in S. pararoseus has not been reported yet. Here, we cloned a cDNA copy of the GGPPS protein-encoding gene crtE from S. pararoseus NGR. The crtE full-length genomic DNA and cDNA are 1,722 and 1,134 bp, respectively, which consist of 9 exons and 8 introns. This gene encodes 377 amino acids protein with a predicted molecular mass of 42.59 kDa and a PI of 5.66. Identification of the crtE gene encoding a functional GGPPS was performed using heterologous complementation detection in Escherichia coli. In vitro enzymatic activity experiments showed that CrtE utilized farnesyl diphosphate (FPP) as an allylic substrate for the condensation reaction with isopentenyl diphosphate (IPP), generating more of the unique product GGPP compared to other allylic substrates. The predicted CrtE 3D-model was analyzed in comparison with yeast GGPPS. The condensation reaction occurs in the cavity of the subunit, and three bulky amino acids (Tyr110, Phe111, and His141) below the cavity prevent further extension of the product. Our findings provide a new source of genes for carotenoid genetic engineering.