Biology subjects

Garza-Garcia, J. J. O.

Publications and source records attributed to Garza-Garcia, J. J. O..

6 recordsLinked to original sources

bioRxiv2026

A cinnamyl alcohol dehydrogenase scaffold organizes monoterpenoid indole alkaloid biosynthesis

Biosynthesis of ~3,000 monoterpenoid indole alkaloids (MIAs), including the anticancer drug vinblastine, involves the highly unstable intermediate strictosidine aglycone. Its formation by strictosidine {beta}-glucosidase (SGD) and subsequent conversion by geissoschizine synthase (GS) occur in spatially separated compartments, representing a major biosynthesis bottleneck. Here we discover VinBLAST, a cinnamyl alcohol dehydrogenase-like protein repurposed as a scaffold for efficient processing of this labile intermediate. VinBLAST physically mediates SGD and GS interaction in the nucleus and allosterically enhances GS catalytic efficiency. VinBLAST homologues from diverse plant families enhance biosynthesis of several representative MIAs, with the production of catharanthine increased to ~160 mg L-1 in yeast, nearly 1,000-fold higher than previous studies. Our discovery provides a missing link in organizing MIA biosynthesis and enables scalable bioproduction of geissoschizine-derived therapeutics.

synthetic biology↗

bioRxiv2025

Ancient gene clusters initiate monoterpene indole alkaloid biosynthesis and C-3 stereochemistry inversion

The inversion of C3 stereochemistry in monoterpenoid indole alkaloids (MIAs), derived from the central precursor strictosidine (3S), is a critical step for the biosynthesis of numerous 3R MIAs and spirooxindoles, including the antihypertensive drug reserpine. While early MIA biosynthesis preserves the 3S configuration, the mechanism underlying C3 inversion has remained unresolved. Here, we identify and biochemically characterize a conserved oxidase-reductase pair in the Gentianales order: the heteroyohimbine/yohimbine/corynanthe C3-oxidase (HYC3O) and C3-reductase (HYC3R), which together invert the 3S stereochemistry to 3R across diverse substrates. Notably, HYC3O and HYC3R reside in gene clusters in Rauvolfia tetraphylla and Catharanthus roseus, homologous to an elusive geissoschizine synthase (GS) cluster we also uncovered. In R. tetraphylla, these clusters are in tandem on a single chromosome, likely derived from segmental duplication, whereas in C. roseus they reside on separate chromosomes due to translocation. Comparative genomics indicate the GS cluster originated at the base of Gentianales ([~]135 Mya), coinciding with the evolution of the strictosidine synthase cluster, while the reserpine cluster arose later in rauvolfioid Apocynaceae. Together, these findings uncover the genomic and biochemical basis for key events in MIA evolution and diversification, providing insights beyond the canonical vinblastine and ajmaline biosynthetic pathways.

biochemistry↗

bioRxiv2024

Stereochemical Insights into Sarpagan and Akuammiline Alkaloid Biosynthesis

The Apocynaceae family produces a diverse array of monoterpenoid indole alkaloids (MIAs) with significant pharmaceutical value. Among these, sarpagan and akuammiline alkaloids stand out for their complex stereochemistry, derived from the enzymatic cyclization and rearrangement of geissoschizine. This study investigates the stereochemical outcomes of sarpagan bridge enzymes (SBEs) and rhazimal synthases (RHS), key players in geissoschizine cyclization and MIA diversification. Using two known and five newly identified enzymes from six plant species, we show that RHS enzymes from Alstonia scholaris, Vinca minor, and Amsonia tabernaemontana exclusively produce the 16R rhazimal stereoisomer. Meanwhile, SBEs from Catharanthus roseus, Tabernaemontana elegans, Vinca minor, and Rauvolfia serpentina likely generate 16R polyneuridine aldehyde; however, downstream aldehyde reductase, deformylase, and esterase activities further epimerize and alter the C16 stereochemistry, yielding naturally occurring alkaloids with distinct C16 stereochemistry across species. These findings, supported by in vitro assays and in planta silencing of C. roseus CrSBE after we reroute biosynthetic flux toward mutated sarpagan MIAs, further reveal enzymatic control over C16 stereochemistry in sarpagan MIA biosynthesis. By elucidating the transformation of diastereomeric intermediates, this work provides key insights into the stereochemical and enzymatic diversification of MIAs in nature.

biochemistry↗

bioRxiv2024

Oxidation of Four Monoterpenoid Indole Alkaloid Classes by Three Cytochrome P450 Monooxygenases from Tabernaemontana litoralis

Cytochrome P450 monooxygenases (CYPs) are well known for their ability to catalyze diverse oxidation reactions, playing a significant role in the biosynthesis of various natural products. In the realm of monoterpenoid indole alkaloids (MIAs), one of the largest groups of alkaloids in nature, CYPs are integral to reactions such as hydroxylation, epoxidation, ring opening, ring rearrangement, and aromatization, contributing to the extensive diversification of these compounds. In this study, we investigate the transcriptome, metabolome, and MIA biosynthesis in Tabernaemontana litoralis (milky way tree), a prolific producer of rare pseudoaspidosperma-type MIAs. Alongside known pseudoaspidosperma biosynthetic genes, we identify and characterize three new CYPs that facilitate regio- and stereospecific oxidation of four MIA skeletons: iboga, aspidosperma, pseudoaspidosperma, and quebrachamine. Notably, the tabersonine 14,15-{beta}-epoxidase catalyzes the formation of pachysiphine, the stereoisomer of 14,15--epoxytabersonine (lochnericine) found in Catharanthus roseus (Madagascar periwinkle) roots. The pseudovincadifformine 18-hydroxylase is the first CYP identified to modify a pseudoaspidosperma skeleton. Additionally, we demonstrate that the enzyme responsible for C10-hydroxylation of the iboga MIA coronaridine also catalyzes the same reaction on voaphylline, which bears a quebrachamine skeleton. With the discovery of a new MIA, 11-hydroxypseudovincadifformine, this study provides a comprehensive understanding of MIA biosynthesis and diversification in T. litoralis, highlighting its potential for further exploration.

biochemistry↗

bioRxiv2022

Biosynthesis of Kratom Opioids

Mitragyna speciosa (kratom) derived monoterpenoid indole alkaloids (MIAs) such as mitragynine and 7-hydroxymitragynine are a new class of opioids with a corynanthe MIA pharmacophore that is responsible for their significantly reduced side effects and superior safety profiles. While botanical kratom has been historically used for stimulation and pain management in Southeast Asia, the biosynthesis of kratom MIAs is not known. In this study, we identified and characterized 9 reductases bearing various degrees of demethyldihydrocoryanthine/demethylcorynantheidine synthase activity and a new SABATH type methyltransferase that catalyzes highly unusual non-aromatic enol methylation from kratom and several other species, which are required in kratom opioids biosynthesis. With unnatural substrate 4-hydroxytryptamine, we further showed the biosynthesis of mitragynine and its epimer speciogynine using these characterized enzymes. The promiscuity of kratom opioid biosynthetic enzymes suggests that derivatives and analogs of kratom opioids may be manufactured in heterologous systems with appropriate enzymes and substrates.

biochemistry↗

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↗