Transcriptome analysis reveals novel enzymes for apo-carotenoid biosynthesis in saffron and allows construction for crocetin synthesis in yeast.

Crocus sativus is generally considered the source of saffron spice and rich in apo-carotenoid compounds such as crocins, crocetin, picrocrocin and safranal, which possess effective pharmacological activities. However, little is known about the exact genes involved in apo-carotenoids biosynthesis in saffron and the potential mechanism of specific accumulation in the stigma. In this study, we integrated different developmental stigmas to perform in-depth transcriptome and dynamic metabolomic analyses to discover the potential key catalytic steps involved in apo-carotenoid biosynthesis in saffron. A total of 61202 unigenes were obtained, and twenty-eight regulators and thirty-two putative carotenogenic genes were captured after the co-expression network analysis. Moreover, 15 candidate genes were predicted to be closely related to safranal and crocin production, in which one aldehyde dehydrogenase (CsALDH3) was validated to oxidize crocetin dialdehyde into crocetin and a crocetin-producing yeast strain was created. In addition, a new branch pathway that catalyses the conversion GGPP to copalol and ent-kaurene by class II diterpene synthases CsCPS1 and three class I diterpene synthases CsEKL1/2/3 were investigated for the first time. Such gene-to-apo-carotenoid landscapes illuminate the synthetic charactersistics and regulators of apo-carotenoid biosynthesis, laying the foundation for deeply understanding the biosynthesis mechanism and metabolic engineering of apo-carotenoids in plant or microbe.

[1]  Xinlei Guo,et al.  Genome-wide characterization and expression analysis of the aldehyde dehydrogenase (ALDH) gene superfamily under abiotic stresses in cotton. , 2017, Gene.

[2]  Huabei Zhang,et al.  Functional Diversification of Kaurene Synthase-Like Genes in Isodon rubescens1[OPEN] , 2017, Plant Physiology.

[3]  Wenhai Xiao,et al.  Heterologous biosynthesis and manipulation of crocetin in Saccharomyces cerevisiae , 2017, Microbial Cell Factories.

[4]  D. Ro,et al.  Biosynthesis of the psychotropic plant diterpene salvinorin A: Discovery and characterization of the Salvia divinorum clerodienyl diphosphate synthase , 2017, The Plant journal : for cell and molecular biology.

[5]  Meirong Jia,et al.  A Pair of Residues That Interactively Affect Diterpene Synthase Product Outcome , 2017, ACS chemical biology.

[6]  Dabing Zhang,et al.  Characterization of factors underlying the metabolic shifts in developing kernels of colored maize , 2016, Scientific Reports.

[7]  Wansheng Chen,et al.  Dynamic metabolic and transcriptomic profiling of methyl jasmonate‐treated hairy roots reveals synthetic characters and regulators of lignan biosynthesis in Isatis indigotica Fort , 2016, Plant biotechnology journal.

[8]  Mukesh Jain,et al.  De novo transcriptome assembly and comprehensive expression profiling in Crocus sativus to gain insights into apocarotenoid biosynthesis , 2016, Scientific Reports.

[9]  Tingting Liu,et al.  Integration of a Decrescent Transcriptome and Metabolomics Dataset of Peucedanum praeruptorum to Investigate the CYP450 and MDR Genes Involved in Coumarins Biosynthesis and Transport , 2015, Front. Plant Sci..

[10]  Liangjiang Wang,et al.  A Genome-Wide Scenario of Terpene Pathways in Self-pollinated Artemisia annua. , 2015, Molecular plant.

[11]  J. Gershenzon,et al.  One amino acid makes the difference: the formation of ent-kaurene and 16α-hydroxy-ent-kaurane by diterpene synthases in poplar , 2015, BMC Plant Biology.

[12]  L. Gómez-Gómez,et al.  Saffron: Its Phytochemistry, Developmental Processes, and Biotechnological Prospects. , 2015, Journal of agricultural and food chemistry.

[13]  Anil Kumar Singh,et al.  Comprehensive transcriptome analysis of Crocus sativus for discovery and expression of genes involved in apocarotenoid biosynthesis , 2015, BMC Genomics.

[14]  J. Mir,et al.  Comparative expression analysis of senescence gene CsNAP and B-class floral development gene CsAP3 during different stages of flower development in Saffron (Crocus sativus L.) , 2015, Physiology and Molecular Biology of Plants.

[15]  Jörg Bohlmann,et al.  Plant diterpene synthases: exploring modularity and metabolic diversity for bioengineering. , 2015, Trends in biotechnology.

[16]  D. Jain,et al.  Identification, cloning and characterization of an ultrapetala transcription factor CsULT1 from Crocus: a novel regulator of apocarotenoid biosynthesis , 2015, BMC Plant Biology.

[17]  C. Starks,et al.  Diterpene synthases of the biosynthetic system of medicinally active diterpenoids in Marrubium vulgare. , 2014, The Plant journal : for cell and molecular biology.

[18]  P. Beyer,et al.  Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis , 2014, Proceedings of the National Academy of Sciences.

[19]  Hideyuki Suzuki,et al.  Integrated Analysis of the Effects of Cold and Dehydration on Rice Metabolites, Phytohormones, and Gene Transcripts1[W][OPEN] , 2014, Plant Physiology.

[20]  Wansheng Chen,et al.  Biosynthesis of the active compounds of Isatis indigotica based on transcriptome sequencing and metabolites profiling , 2013, BMC Genomics.

[21]  S. Al‐Babili,et al.  A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments , 2013, Journal of experimental botany.

[22]  J. Bohlmann,et al.  Gene Discovery of Modular Diterpene Metabolism in Nonmodel Systems1[W][OA] , 2013, Plant Physiology.

[23]  N. Darzentas,et al.  The study of the E-class SEPALLATA3-like MADS-box genes in wild-type and mutant flowers of cultivated saffron crocus (Crocus sativus L.) and its putative progenitors. , 2011, Journal of plant physiology.

[24]  S. Akhondzadeh,et al.  Cardiovascular Effects of Saffron: An Evidence-Based Review , 2011, The journal of Tehran Heart Center.

[25]  F. Kaplan,et al.  Identity, regulation, and activity of inducible diterpenoid phytoalexins in maize , 2011, Proceedings of the National Academy of Sciences.

[26]  B. Pogson,et al.  Source to sink: regulation of carotenoid biosynthesis in plants. , 2010, Trends in plant science.

[27]  Dana J Morrone,et al.  Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering , 2009, Applied Microbiology and Biotechnology.

[28]  H. Weiner,et al.  Involvement of snapdragon benzaldehyde dehydrogenase in benzoic acid biosynthesis. , 2009, The Plant journal : for cell and molecular biology.

[29]  A. Granell,et al.  Metabolite and target transcript analyses during Crocus sativus stigma development. , 2009, Phytochemistry.

[30]  M. Rodrigo,et al.  Molecular and functional characterization of a novel chromoplast-specific lycopene β-cyclase from Citrus and its relation to lycopene accumulation , 2009, Journal of experimental botany.

[31]  P. Tarantilis,et al.  Effects of the active constituents of Crocus sativus L., crocins, in an animal model of anxiety. , 2008, Phytomedicine : international journal of phytotherapy and phytopharmacology.

[32]  A. Granell,et al.  Cytosolic and Plastoglobule-targeted Carotenoid Dioxygenases from Crocus sativus Are Both Involved in β-Ionone Release* , 2008, Journal of Biological Chemistry.

[33]  Yongsheng Liu,et al.  Significant improvement of stress tolerance in tobacco plants by overexpressing a stress-responsive aldehyde dehydrogenase gene from maize (Zea mays) , 2008, Plant Molecular Biology.

[34]  W. Scheible,et al.  Eleven Golden Rules of Quantitative RT-PCR , 2008, The Plant Cell Online.

[35]  W. Schwab,et al.  Biosynthesis of plant-derived flavor compounds. , 2008, The Plant journal : for cell and molecular biology.

[36]  Dana J Morrone,et al.  Increasing complexity of a diterpene synthase reaction with a single residue switch. , 2008, Journal of the American Chemical Society.

[37]  P. Beyer,et al.  A Third Phytoene Synthase Is Devoted to Abiotic Stress-Induced Abscisic Acid Formation in Rice and Defines Functional Diversification of Phytoene Synthase Genes1[W] , 2008, Plant Physiology.

[38]  N. D’Agostino,et al.  An EST database from saffron stigmas , 2007, BMC Plant Biology.

[39]  Faqiang Li,et al.  Maize Y9 Encodes a Product Essential for 15-cis-ζ-Carotene Isomerization1[OA] , 2007, Plant Physiology.

[40]  A. Kalivas,et al.  Heterotopic expression of B-class floral homeotic genes PISTILLATA/GLOBOSA supports a modified model for crocus (Crocus sativus L.) flower formation , 2007, DNA sequence : the journal of DNA sequencing and mapping.

[41]  Meimei Xu,et al.  The Maize An2 Gene is Induced by Fusarium Attack and Encodes an ent-Copalyl Diphosphate Synthase , 2005, Plant Molecular Biology.

[42]  J. Fernández,et al.  Implications of Carotenoid Biosynthetic Genes in Apocarotenoid Formation during the Stigma Development of Crocus sativus and Its Closer Relatives1 , 2005, Plant Physiology.

[43]  L. Gómez-Gómez,et al.  Glucosylation of the saffron apocarotenoid crocetin by a glucosyltransferase isolated from Crocus sativus stigmas , 2004, Planta.

[44]  P. Matthews,et al.  Maize phytoene desaturase and zeta-carotene desaturase catalyse a poly-Z desaturation pathway: implications for genetic engineering of carotenoid content among cereal crops. , 2003, Journal of experimental botany.

[45]  B. Camara,et al.  Biosynthesis of the Food and Cosmetic Plant Pigment Bixin (Annatto) , 2003, Science.

[46]  L. Tang,et al.  Increased crocin production and induction frequency of stigma-like-structure from floral organs of Crocus sativus L. by precursor feeding , 2003, Plant Cell, Tissue and Organ Culture.

[47]  R. Peters,et al.  Abietadiene synthase catalysis: conserved residues involved in protonation-initiated cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate. , 2002, Biochemistry.

[48]  P M Bramley,et al.  Application of high‐performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids , 2000 .

[49]  D. Zamir,et al.  An alternative pathway to beta -carotene formation in plant chromoplasts discovered by map-based cloning of beta and old-gold color mutations in tomato. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[50]  M. Díaz-Guerra,et al.  In vitro activation of macrophages by a novel proteoglycan isolated from corms of Crocus sativus L. , 1999, Cancer letters.

[51]  P. Christou,et al.  The carotenoid cleavage dioxygenase CCD2 catalysing the synthesis of crocetin in spring crocuses and saffron is a plastidial enzyme. , 2016, The New phytologist.

[52]  C. Zheng,et al.  Antidepressant properties of bioactive fractions from the extract of Crocus sativus L. , 2009, Journal of Natural Medicines.

[53]  Rogelio Pereda-Miranda,et al.  HPLC quantification of major active components from 11 different saffron (Crocus sativus L.) sources , 2007 .