Functional divergence of CYP76AKs shapes the chemodiversity of abietane-type diterpenoids in genus Salvia

Hu, Jiadong; Qiu, Shi; Wang, Feiyan; Li, Qing; Xiang, Chun‐Lei; Peng, Di; Wu, Ziding; Jiang, Rui; Li, Jinxing; Zeng, Zhen; Wang, Jing; Wang, Xingxing; Zhang, Yuchen; Fang, Shiyuan; Qiao, Yu; Ding, Jie; Jiang, Yun; Xu, Zhichao; Chen, J.-L.; Chen, Wansheng

doi:10.1038/s41467-023-40401-y

Cited by 8 publications

(6 citation statements)

References 95 publications

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“…Protein expression was then induced with yeast extract peptone dextrose medium with 2% galactose for 16 hour at 28°C and 200 rpm. Then, the culture was immediately used for microsomal protein isolation following previous instructions ( 102 ). Enzyme reactions with CYP-enriched microsomal proteins were performed in tris-HCl buffer (pH 7.5) containing 5 μg of microsomal protein, 500 μM NADPH (reduced form of nicotinamide adenine dinucleotide phosphate), 50 μM senkyunolide A or ligustilide, and NADPH Regeneration System (Promega) in a total reaction volume of 200 μl.…”

Section: Methodsmentioning

confidence: 99%

Haplotype-phased genome unveils the butylphthalide biosynthesis and homoploid hybrid origin of Ligusticum chuanxiong

Nie,

Chen,

Hou

et al. 2024

Sci. Adv.

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Butylphthalide is one of the first-line drugs for ischemic stroke therapy, while no biosynthetic enzyme for butylphthalide has been reported. Here, we present a haplotype-resolved genome of Ligusticum chuanxiong , a long-cultivated and phthalide-rich medicinal plant in Apiaceae. On the basis of comprehensive screening, four Fe(II)- and 2-oxoglutarate–dependent dioxygenases and two CYPs were mined and further biochemically verified as phthalide C-4/C-5 desaturases (P4,5Ds) that effectively promoted the forming of ( S )-3- n -butylphthalide and butylidenephthalide. The substrate promiscuity and functional redundancy featured for P4,5Ds may contribute to the high phthalide diversity in L. chuanxiong . Notably, comparative genomic evidence supported L. chuanxiong as a homoploid hybrid with Ligusticum sinense as a potential parent. The two haplotypes demonstrated exceptional structure variance and diverged around 3.42 million years ago. Our study is an icebreaker for the dissection of phthalide biosynthetic pathway and reveals the hybrid origin of L. chuanxiong , which will facilitate the metabolic engineering for ( S )-3- n -butylphthalide production and breeding for L. chuanxiong .

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Section: Methodsmentioning

confidence: 99%

Haplotype-phased genome unveils the butylphthalide biosynthesis and homoploid hybrid origin of Ligusticum chuanxiong

Nie,

Chen,

Hou

et al. 2024

Sci. Adv.

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“…The products of the catalytic reaction and the extracts of the different plant species samples were analysed using an ACQUITY UPLC system coupled to a Xevo G2-XS QToF mass spectrometer (Waters), which was equipped with an electrospray ionization source. The conditions for the UPLC-Q-TOF/MS analysis were set as previously described (Hu et al, 2023). Mass-to-charge ratio (m/z) values are listed in Table S2.…”

Section: Metabolomic Profiling and Enzyme Catalytic Assays Analysismentioning

confidence: 99%

Versatile CYP98A enzymes catalyse meta‐hydroxylation reveals diversity of salvianolic acids biosynthesis

Zhou,

Feng,

Huo

et al. 2024

Plant Biotechnology Journal

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SummarySalvianolic acids (SA), such as rosmarinic acid (RA), danshensu (DSS), and their derivative salvianolic acid B (SAB), etc. widely existed in Lamiaceae and Boraginaceae families, are of interest due to medicinal properties in the pharmaceutical industries. Hundreds of studies in past decades described that 4‐coumaroyl‐CoA and 4‐hydroxyphenyllactic acid (4‐HPL) are common substrates to biosynthesize SA with participation of rosmarinic acid synthase (RAS) and cytochrome P450 98A (CYP98A) subfamily enzymes in different plants. However, in our recent study, several acyl donors and acceptors included DSS as well as their ester‐forming products all were determined in SA‐rich plants, which indicated that previous recognition to SA biosynthesis is insufficient. Here, we used Salvia miltiorrhiza, a representative important medicinal plant rich in SA, to elucidate the diversity of SA biosynthesis. Various acyl donors as well as acceptors are catalysed by SmRAS to form precursors of RA and two SmCYP98A family members, SmCYP98A14 and SmCYP98A75, are responsible for different positions' meta‐hydroxylation of these precursors. SmCYP98A75 preferentially catalyses C‐3′ hydroxylation, and SmCYP98A14 preferentially catalyses C‐3 hydroxylation in RA generation. In addition, relative to C‐3′ hydroxylation of the acyl acceptor moiety in RA biosynthesis, SmCYP98A75 has been verified as the first enzyme that participates in DSS formation. Furthermore, SmCYP98A enzymes knockout resulted in the decrease and overexpression leaded to dramatic increase of SA accumlation. Our study provides new insights into SA biosynthesis diversity in SA‐abundant species and versatility of CYP98A enzymes catalytic preference in meta‐hydroxylation reactions. Moreover, CYP98A enzymes are ideal metabolic engineering targets to elevate SA content.

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“…Gene duplication played an important role in the expanding of gene families in plant genomes [18,19,42]. It has been demonstrated that, multiple gene duplication events at the family level or above in campanulids, resulted in a large number of FAD2 homologs in Asterales and Apiales [7].…”

Section: Characteristics Of Fad2 Family Genes In Campanulidsmentioning

confidence: 99%

“…4, Table 1). After gene duplication, plants take up eliminating most redundant gene copies in a long evolutionary process generally, with some duplicated copies retained via neofunctionalizations, which is the cause of the occurrence of numerous specialized metabolites in some lineage-speci c plants [7,18,19,43]. Therefore, it can be concluded that gene duplication followed by functional diversi cation of FAD2 genes, giving rise to diverse PAs in campanulids.…”

Section: Characteristics Of Fad2 Family Genes In Campanulidsmentioning

confidence: 99%

“…Previous studies exploring the emergence of various lineage-speci c specialized metabolites, have revealed that gene duplication and mutations, and the variations in the number of gene family members followed by functional diversi cation, is of central importance to this process [17,18,19]. Interestingly, FAD2 gene family has been found to expand extensively in Asterales and Apiales [7].…”

Section: Introductionmentioning

confidence: 99%

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Comprehensive evolutionary analysis of the FAD2 gene family in representatives of campanulids: identification and characterization of candidate genes from the plant polyacetylene metabolism

Cao,

Wang,

Liu

et al. 2024

Preprint

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Background Polyacetylenes (PAs) are a kind of specialized plant defense compounds that mainly occur in campanulid plants. Fatty Acid Desaturase 2 (FAD2) catalyzes desaturation in the initial stage of PA biosynthesis, and tends to be involved in later steps of oxidation reactions as well. The FAD2 enzymes not only have desaturase activity, but can also perform other fatty acid modifications, giving rise to various unusual fatty acids, including the precursors to PAs.Methods and results We present a genome-wide survey of FAD2 genes in nine species, covering all main families of PA source in campanulids and the model plant Solanum lycopersicum, and reconstruct the evolutionary history of this gene family. We uncovered the high conservatism of motif composition, gene structural characteristics and expression patterns of the FAD2 members, in spite of their functional diversification. The number of genes expanded extensively in Asterales and Apiales, two main clades of campanulids, by various gene duplication.Conclusion We provided a list of FAD2 candidates that potentially underlying the biosynthesis of PAs using an approach integrating the phylogenetic framework and molecular evolution rates studies. This work will significantly broaden our perspective on the functional diversity of FAD2 enzymes, and lay a foundation for subsequent functional analysis aimed at discovering further FAD2 genes involved in PA biosynthesis.

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Functional divergence of CYP76AKs shapes the chemodiversity of abietane-type diterpenoids in genus Salvia

Cited by 8 publications

References 95 publications

Haplotype-phased genome unveils the butylphthalide biosynthesis and homoploid hybrid origin of Ligusticum chuanxiong

Haplotype-phased genome unveils the butylphthalide biosynthesis and homoploid hybrid origin of Ligusticum chuanxiong

Versatile CYP98A enzymes catalyse meta‐hydroxylation reveals diversity of salvianolic acids biosynthesis

Comprehensive evolutionary analysis of the FAD2 gene family in representatives of campanulids: identification and characterization of candidate genes from the plant polyacetylene metabolism

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