Biology subjects

Ro, D.-K.

Publications and source records attributed to Ro, D.-K..

4 recordsLinked to original sources

bioRxiv2024

A chromosome level reference genome of Diviners sage (Salvia divinorum) provides insight into salvinorin A biosynthesis

BackgroundDiviners sage (Salvia divinorum; Lamiaceae) is the source of the powerful hallucinogen salvinorin A (SalA). This neoclerodane diterpenoid is an agonist of the human K opioid receptor with potential medical applications in the treatment of chronic pain, addiction, and post-traumatic stress disorder. Only two steps of the approximately twelve step biosynthetic sequence leading to SalA have been resolved to date. ResultsTo facilitate pathway elucidation in this ethnomedicinal plant species, here we report a chromosome level genome assembly. A high-quality genome sequence was assembled with an N50 value of 41.4 Mb and a BUSCO completeness score of 98.4%. The diploid (2n = 22) genome of [~]541 Mb is comparable in size and ploidy to most other members of this genus. Two diterpene biosynthetic gene clusters were identified and are highly enriched in previously unidentified cytochrome P450s as well as crotonolide G synthase, which forms the dihydrofuran ring early in the SalA pathway. Coding sequences for other enzyme classes with likely involvement in downstream steps of the SalA pathway (BAHD acyl transferases, alcohol dehydrogenases, and O-methyl transferases) were scattered throughout the genome with no clear indication of clustering. Differential gene expression analysis suggests that most of these genes are not inducible by methyl jasmonate treatment. ConclusionsThis genome sequence and associated gene annotation are among the highest resolution in Salvia, a genus well known for the medicinal properties of its members. Here we have identified the cohort of genes responsible for the remaining steps in the SalA pathway. This genome sequence and associated candidate genes will facilitate the elucidation of SalA biosynthesis and enable an exploration of its full clinical potential.

genomics↗

bioRxiv2022

Improved protein glycosylation enabled heterologous biosynthesis of monoterpenoid indole alkaloids and their unnatural derivatives in yeast

With over 3,000 reported structures, monoterpenoid indole alkaloids (MIAs) constitute one of the largest alkaloid groups in nature, including the clinically important anticancer drug vinblastine and its semi-synthetic derivatives from Catharanthus roseus (Madagascars periwinkle). With the elucidation of the complete 28-step biosynthesis for anhydrovinblastine, it is possible to investigate the heterologous production of vinblastine and other medicinal MIAs. In this study, we successfully expressed the flavoenzyme O-acetylstemmadenine oxidase in Saccharomyces cerevisiae (bakers yeast) by signal peptide modification, which is a vinblastine biosynthetic gene that has not been functionally expressed in this system. We also report the simultaneous genomic integration of [~]18 kb MIA biosynthetic gene cassettes as single copies by CRISPR-Cas9 in bakers yeast, which enabled the biosynthesis of vinblastine precursors catharanthine and tabersonine from the feedstocks secologanin and tryptamine. We further demonstrated the biosynthesis of fluorinated and hydroxylated catharanthine and tabersonine derivatives using our yeasts, which showed that the MIA biosynthesis accommodates unnatural substrates, and the system can be further explored to produce other complex MIAs. With over 3,000 members, monoterpenoid indole alkaloids (MIA) are one of the largest and most diverse alkaloids in nature including many human medicines, such as chemotherapeutics vinblastine from Catharanthus roseus (Madagascars periwinkle) and camptothecin from Camptotheca accuminata (happy tree), and antiarrhythmic ajmaline from Rauwolfia serpentina (Indian snakeroot).1 Recent studies have elucidated the complete 28-step biosynthetic pathway for anhydrovinblastine in C. roseus, which involves diverting a primary monoterpene geranyl pyrophosphate into the biosynthesis of secologanin via the iridoid pathway (9 steps), genesis of the first MIA strictosidine that is the universal precursor to almost all MIAs (2 steps), conversion of strictosidine to iboga type MIA catharanthine and aspidosperma type tabersonine (9 steps), decorating tabersonine to vindoline (7 steps), and the final step that couples vindoline and catharanthine to make anhydrovinblastine (Fig. 1). 2-12 These studies not only revealed the remarkable complexity of MIA formations but also enabled the exploration in heterologous production of bioactive MIAs and intermediates that are usually found in low quantities in their natural sources. Notably, strictosidine and a related corynanthe type MIA ajmalicine have been produced de novo in Saccharomyces cerevisiae (bakers yeast), 13,14 while vindoline has been produced in bakers yeast from tabersonine feedstock. 3,15,16 For strictosidine production in yeast, the challenges lie in the generally low monoterpene biosynthesis output and the intermediates consumption by yeast native metabolism.13,14,17 While studies did not report rapid MIA consumption by yeast, vindoline yields were improved by optimizing the stoichiometry of cytochrome P450 monooxygenase (CYP), CYP redox partner CYP reductase (CPR), and other factors related with CYP activities such as endoplasmic reticulum (ER) homeostasis and NADPH co-factor regeneration that are commonly exploited.15,16 In this study, we constructed yeast strains containing the remaining vinblastine biosynthetic segment and produced catharanthine and tabersonine by feeding precursors, secologanin and tryptamine, as well as their unnatural derivatives by feeding substituted tryptamine. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/495323v1_fig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@52eda3org.highwire.dtl.DTLVardef@6f8d6dorg.highwire.dtl.DTLVardef@1ae0e1aorg.highwire.dtl.DTLVardef@1fed9f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO The biosynthetic pathway for monoterpenoid indole alkaloids (MIAs) catharanthine and vindoline in C. roseus, which couple to form the anticancer anhydrovinblastine and other derivatives. TDC: tryptophan decarboxylase (Genbank P17770); STR: strictosidine synthase (Genbank CAA43936); SGD: strictosidine -glucosidase (Genbank AAF28800); GS: geissoschizine synthase (Genbank MF770507); GO: geissoschizine oxidase (Genbank MF770508); Redox1/2: oxidized geissoschizine reductase 1/2 (Genbank MF770509, MF770510); SAT: stemmadenine O-acetyltransferase (Genbank MF770511); ASO: O-acetylstemmadenine oxidase (Genbank MH136588); DPAS: dihydroprecondylocarpine synthase (Genbank A0A1B1FHP3); HL1: hydrolase 1/catharanthine synthase (Genbank MF770512); HL2: hydrolase 2/tabersonine synthase (Genbank MF770513). C_FIG

synthetic biology↗

bioRxiv2021

The Taxus genome provides insights into paclitaxel biosynthesis

The ancient gymnosperm genus Taxus is the exclusive source of the anticancer drug paclitaxel, yet no reference genome sequences are available for comprehensively elucidating the paclitaxel biosynthesis pathway. We have completed a chromosome-level genome of Taxus chinensis var. mairei with a total length of 10.23 Gb. Taxus shared an ancestral whole-genome duplication with the coniferophyte lineage and underwent distinct transposon evolution. We discovered a unique physical and functional grouping of CYP725As (cytochrome P450) in the Taxus genome for paclitaxel biosynthesis. We also identified a gene cluster in the taxadiene biosynthesis, which was mainly formed by gene duplications. This study will facilitate the elucidation of paclitaxel biosynthesis and unleash the biotechnological potential of Taxus. One Sentence SummaryA chromosome-level genome assembly of Taxus chinensis var. mairei uncovers its unique genome evolution process and genetic architectures for the paclitaxel biosynthesis pathway.

genomics↗

bioRxiv2018

Characterization of cis-prenyltransferase complexes in guayule (Parthenium argentatum), an alternative natural rubber-producing plant

Guayule (Parthenium argentatum) is a perennial shrub in the Asteraceae family and synthesizes a high quality, hypoallergenic cis-1,4-polyisoprene (or natural rubber; NR). Despite its potential to be an alternative NR supplier, the enzymes for cis-polyisoprene biosynthesis have not been comprehensively studied in guayule. Recently, implications of the protein complex involving cis-prenyltransferases (CPTs) and CPT-binding proteins (CBPs) in NR biosynthesis were shown in lettuce and dandelion, but such protein complexes have yet to be examined in guayule. Here we identified four guayule genes - three PaCPTs (PaCPT1-3) and one PaCBP, whose protein products form PaCPT/PaCBP complexes. Co-expression of both PaCBP and each of the PaCPTs could complemented the dolichol (a short cis-polyisoprene)-deficient yeast, whereas the individual expressions could not. Microsomes from the PaCPT/PaCBP-expressing yeast efficiently incorporated 14C-isopentenyl diphosphate into dehydrodolichyl diphosphates. Furthermore, co-immunoprecipitation and split-ubiquitin yeast 2-hybrid assays using PaCPTs and PaCBP confirmed the formation of protein complexes. Of the three PaCPTs, transcriptomics analysis indicated that the protein complex formed by PaCPT3 and PaCBP is likely to be the key component in guayule NR biosynthesis. The comprehensive analyses of these PaCPTs and PaCBP here provide the foundational knowledge to generate a high NR-yielding guayule.

plant biology↗