The Amaryllidaceae alkaloids are a family of amino acid derived alkaloids with many biological activities; examples include haemanthamine, haemanthidine, galanthamine, lycorine, and maritidine. ...Central to the biosynthesis of the majority of these alkaloids is a C-C phenol-coupling reaction that can have para-para', para-ortho', or ortho-para' regiospecificity. Through comparative transcriptomics of Narcissus sp. aff. pseudonarcissus, Galanthus sp., and Galanthus elwesii we have identified a para-para' C-C phenol coupling cytochrome P450, CYP96T1, capable of forming the products (10bR,4aS)-noroxomaritidine and (10bS,4aR)-noroxomaritidine from 4'-O-methylnorbelladine. CYP96T1 was also shown to catalyzed formation of the para-ortho' phenol coupled product, N-demethylnarwedine, as less than 1% of the total product. CYP96T1 co-expresses with the previously characterized norbelladine 4'-O-methyltransferase. The discovery of CYP96T1 is of special interest because it catalyzes the first major branch in Amaryllidaceae alkaloid biosynthesis. CYP96T1 is also the first phenol-coupling enzyme characterized from a monocot.
Summary
Steroid alkaloids have been shown to elicit a wide range of pharmacological effects that include anticancer and antifungal activities. Understanding the biosynthesis of these molecules is ...essential to bioengineering for sustainable production. Herein, we investigate the biosynthetic pathway to cyclopamine, a steroid alkaloid that shows promising antineoplastic activities. Supply of cyclopamine is limited, as the current source is solely derived from wild collection of the plant Veratrum californicum. To elucidate the early stages of the pathway to cyclopamine, we interrogated a V. californicum RNA‐seq dataset using the cyclopamine accumulation profile as a predefined model for gene expression with the pattern‐matching algorithm Haystack. Refactoring candidate genes in Sf9 insect cells led to discovery of four enzymes that catalyze the first six steps in steroid alkaloid biosynthesis to produce verazine, a predicted precursor to cyclopamine. Three of the enzymes are cytochromes P450 while the fourth is a γ‐aminobutyrate transaminase; together they produce verazine from cholesterol.
Significance Statement
The first four enzymes involved in the biosynthesis of the steroid alkaloid verazine, a predicted precursor to the antineoplastic cyclopamine in Veratrum californicum, were discovered: cholesterol 22‐hydroxylase, 22‐hydroxycholesterol 26‐hydroxylase/oxidase, 22‐hydroxycholesterol‐26‐al transaminase, and 22‐hydroxy‐26‐aminocholesterol 22‐oxidase. The pathway to verazine has been refactored in Spodoptera frugiperda Sf9 cells.
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►
Argemone mexicana EST dataset created from alkaloid-rich root tissue. ► Unigenes with homology to alkaloid biosynthesis genes identified. ► CYP719A14 newly assigned as ...cheilanthifoline synthase. ► CYP719A13 is homolog of
Eschscholzia californica stylopine synthase. ► CYP719A13 participates in both sanguinarine and berberine biosynthesis.
Formation of the methylenedioxy bridge is an integral step in the biosynthesis of benzo
cphenanthridine and protoberberine alkaloids in the Papaveraceae family of plants. This reaction in plants is catalyzed by cytochrome P450-dependent enzymes. Two cDNAs that encode cytochrome P450 enzymes belonging to the CYP719 family were identified upon interrogation of an EST dataset prepared from 2-month-old plantlets of the Mexican prickly poppy
Argemone mexicana that accumulated the benzo
cphenanthridine alkaloid sanguinarine and the protoberberine alkaloid berberine. CYP719A13 and CYP719A14 are 58% identical to each other and 77% and 60% identical, respectively, to stylopine synthase CYP719A2 of benzo
cphenanthridine biosynthesis in
Eschscholzia californica. Functional heterologous expression of
CYP719A14 and
CYP719A13 in
Spodoptera frugiperda Sf9 cells produced recombinant enzymes that catalyzed the formation of the methylenedioxy bridge of (
S)-cheilanthifoline from (
S)-scoulerine and of (
S)-stylopine from (
S)-cheilanthifoline, respectively. Twenty-seven potential substrates were tested with each enzyme. Whereas CYP719A14 transformed only (
S)-scoulerine to (
S)-cheilanthifoline (
K
m
1.9
±
0.3;
k
cat
/
K
m
1.7), CYP719A13 converted (
S)-tetrahydrocolumbamine to (
S)-canadine (
K
m
2.7
±
1.3;
k
cat
/
K
m
12.8), (
S)-cheilanthifoline to (
S)-stylopine (
K
m
5.2
±
3.0;
k
cat
/
K
m
2.6) and (
S)-scoulerine to (
S)-nandinine (
K
m
8.1
±
1.9;
k
cat
/
K
m
0.7). These results indicate that although CYP719A14 participates in only sanguinarine biosynthesis, CYP719A13 can be involved in both sanguinarine
and berberine formation in
A. mexicana.
Main conclusion Camelina was bioengineered to accumulate (4S)-limonene and (+)-δ-cadinene in seed. Plastidic localization of the recombinant enzymes resulted in higher yields than cytosolic ...localization. Overexpressing 1-deoxy-D-xylulose-5-phosphate synthase (DXS) further increased terpene accumulation. Many plant-derived compounds of high value for industrial or pharmaceutical applications originate from plant species that are not amenable to cultivation. Biotechnological production in low-input organisms is an attractive alternative. Several microbes are well established as biotechnological production platforms; however, their growth requires fermentation units, energy input, and nutrients. Plant-based production systems potentially allow the generation of high-value compounds on arable land with minimal input. Here we explore whether Camelina sativa (camelina), an emerging low-input non-foodstuff Brassicaceae oilseed crop grown on marginal lands or as a rotation crop on fallow land, can successfully be refactored to produce and store novel compounds in seed. As proof-of-concept, we use the cyclic monoterpene hydrocarbon (4S)-limonene and the bicyclic sesquiterpene hydrocarbon (+)-δ-cadinene, which have potential biofuel and industrial solvent applications. Post-translational translocation of the recombinant enzymes to the plastid with concurrent overexpression of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) resulted in the accumulation of (4S)-limonene and (+)-δ-cadinene up to 7 mg g⁻¹ seed and 5 mg g⁻¹ seed, respectively. This study presents the framework for rapid engineering of camelina oilseed production platforms for terpene-based high-value compounds.
Summary
S‐Adenosyl‐l‐methionine:(R,S)‐reticuline 7‐O‐methyltransferase converts reticuline to laudanine in tetrahydrobenzylisoquinoline biosynthesis in the opium poppy Papaver somniferum. This enzyme ...activity has not yet been detected in plants. A proteomic analysis of P. somniferum latex identified a gel spot that contained a protein(s) whose partial amino acid sequences were homologous to those of plant O‐methyltransferases. cDNA was amplified from P. somniferum RNA by reverse transcription PCR using primers based on these internal amino acid sequences. Recombinant protein was then expressed in Spodoptera frugiperda Sf9 cells in a baculovirus expression vector. Steady‐state kinetic measurements with one heterologously expressed enzyme and mass spectrometric analysis of the enzymatic products suggested that this unusual enzyme is capable of carrying through sequential O‐methylations on the isoquinoline and on the benzyl moiety of several substrates. The tetrahydrobenzylisoquinolines (R)‐reticuline (4.2 sec−1 mm−1), (S)‐reticuline (4.5 sec−1 mm−1), (R)‐protosinomenine (1.7 sec−1 mm−1), and (R,S)‐isoorientaline (1.4 sec−1 mm−1) as well as guaiacol (5.9 sec−1 mm−1) and isovanillic acid (1.2 sec−1 mm−1) are O‐methylated by the enzyme with the ratio kcat/K m shown in parentheses. A P. somniferum cDNA encoding (R,S)‐norcoclaurine 6‐O‐methyltransferase was similarly isolated and characterized. This enzyme was less permissive, methylating only (R,S)‐norcoclaurine (7.4 sec−1 mm−1), (R)‐norprotosinomenine (4.1 sec−1 mm−1), (S)‐norprotosinomenine (4.0 sec−1 mm−1) and (R,S)‐isoorientaline (1.0 sec−1 mm−1). A phylogenetic comparison of the amino acid sequences of these O‐methyltransferases to those from 28 other plant species suggests that these enzymes group more closely to isoquinoline biosynthetic O‐methyltransferases from Coptis japonica than to those from Thalictrum tuberosum that can O‐methylate both alkaloid and phenylpropanoid substrates.
The flame lily
,
Gloriosa superba
L., is one of the two primary sources of the anti-inflammatory drug, colchicine. Previous studies have shown that a higher level of colchicine production occurs in ...the rhizomes than in leaves and roots. Earlier precursor feeding and transcriptome analysis of
G. superba
have provided a putative pathway and candidate genes involved in colchicine biosynthesis. Comparative analysis of expression levels of candidate pathway genes in different tissues of
G. superba
using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) can reveal highly expressed genes in the rhizome compared to other tissues which could suggest roles of the gene products in colchicine biosynthesis. Normalization is an important step in effectively analyzing differential gene expression by qRT-PCR with broader applications. The current study selected candidate reference genes from the transcriptome datasets and analyzed them to determine the most stable genes for normalization of colchicine biosynthesis-related genes. Using RefFinder, one stable reference gene, UBC22, was selected to normalize gene expression levels of candidate methyltransferase (MT) genes in the leaves, roots, and rhizomes of
G. superba
. With UBC22 as reference gene, the methyltransferases, GsOMT1, GsOMT3, and GsOMT4 showed significantly higher expression levels in the rhizome of
G. superba
, while MT31794 was more highly expressed in the roots. In conclusion, the current results showed a viable reference gene expression analysis system that could help elucidate colchicine biosynthesis and its exploitation for increased production of the drug in
G. superba
.
Berberine bridge enzyme (BBE) is involved in the transformation of (S)-reticuline to (S)-scoulerine in benzophenanthridine alkaloid biosynthesis of plants. In this report, we describe the high level ...expression of BBE encoded by the gene from Eschscholzia californica (California poppy) in the methylotrophic yeast Pichia pastoris employing the secretory pathway of the host organism. Using a two-step chromatographic purification protocol, 120 mg of BBE could be obtained from 1 liter of fermentation culture. The purified protein exhibits a turnover number for substrate conversion of 8.2 s-1. The recombinant enzyme is glycosylated and carries a covalently attached FAD cofactor. In addition to the previously known covalent attachment of the 8α-position of the flavin ring system to a histidine (His-104), we could also demonstrate that a covalent linkage between the 6-position and a thiol group of a cysteine residue (Cys-166) is present in BBE. The major evidence for the occurrence of a bi-covalently attached FAD cofactor is provided by N-terminal amino acid sequencing and mass spectrometric analysis of the isolated flavin-containing peptide. Furthermore, it could be shown that anaerobic photoirradiation leads to cleavage of the linkage between the 6-cysteinyl group yielding 6-mercaptoflavin and a peptide with the cysteine residue replaced by alanine due to breakage of the C-S bond. Overall, BBE is shown to exhibit typical flavoprotein oxidase properties as exemplified by the occurrence of an anionic flavin semiquinone species and formation of a flavin N(5)-sulfite adduct.
Protein phosphorylation is one of the most prevalent posttranslational modifications found in eukaryotic systems. It serves as a key molecular mechanism that regulates protein function in response to ...environmental stimuli. The Mut9-like kinases (MLKs) are a plant-specific family of Ser/Thr kinases linked to light, circadian, and abiotic stress signaling. Here we use quantitative phosphoproteomics in conjunction with global proteomic analysis to explore the role of the MLKs in daily protein dynamics. Proteins involved in light, circadian, and hormone signaling, as well as several chromatin-modifying enzymes and DNA damage response factors, were found to have altered phosphorylation profiles in the absence of MLK family kinases. In addition to altered phosphorylation levels, mlk mutant seedlings have an increase in glucosinolate metabolism enzymes. Subsequently, we show that a functional consequence of the changes to the proteome and phosphoproteome in mlk mutant plants is elevated glucosinolate accumulation and increased sensitivity to DNA damaging agents. Combined with previous reports, this work supports the involvement of MLKs in a diverse set of stress responses and developmental processes, suggesting that the MLKs serve as key regulators linking environmental inputs to developmental outputs.
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•MUT9-LIKE KINASE mutant quantitative proteome and phosphoproteome measured.•Changes to proteome and phosphoproteome are specific to genotype and environment.•Loss of MLKs alters glucosinolate enzyme abundance and metabolism.•Loss of MLKs increases plant sensitivity to UV radiation and DNA damage agents.
The MUT9-like kinases are a family of plant-specific nuclear-localized kinases with roles in diverse signaling pathways, including light sensing, phytohormone perception, and the circadian clock. The proteome and phosphoproteome of compound mlk mutant seedlings have been determined under light and dark conditions. These experiments identify new roles for these kinases regulating secondary plant metabolism and stress responses, tested through metabolite analysis and assaying seedling sensitivity to DNA damaging agents.
Berberine bridge enzyme catalyzes the conversion of (S)-reticuline to (S)-scoulerine by formation of a carbon-carbon bond between the N-methyl group and the phenolic ring. We elucidated the structure ...of berberine bridge enzyme from Eschscholzia californica and determined the kinetic rates for three active site protein variants. Here we propose a catalytic mechanism combining base-catalyzed proton abstraction with concerted carbon-carbon coupling accompanied by hydride transfer from the N-methyl group to the N5 atom of the FAD cofactor.
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