Plug-and-Play Benzylisoquinoline Alkaloid Biosynthetic Gene Discovery in Engineered Yeast

Morris, Jeremy S.; Dastmalchi, Mehran; Li, J.; Chang, Lihong; Chen, X.; Hagel, Jillian M.; Facchini, Peter J.

doi:10.1016/bs.mie.2016.03.023

Cited by 14 publications

(18 citation statements)

References 88 publications

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“…Perhaps due to uncertainty surrounding their mechanisms of action, PR10/Bet v1‐like proteins have not yet been employed in heterologous flavonoid biosynthesis. Rather than waiting for these fundamental questions to be answered, we suggest that a plug‐and‐play approach could immediately be used to screen family members for relevant properties in existing flavonoid‐engineered microorganisms [52,165] . Given the often nonspecific properties of this protein family, members may even find application in heterologous pathways for the biosynthesis of unnatural molecules, as recently demonstrated for hydrocodone.…”

Section: Discussionmentioning

confidence: 99%

PR10/Bet v1‐like Proteins as Novel Contributors to Plant Biochemical Diversity

et al. 2020

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Pathogenesis‐related (PR) proteins constitute a broad class of plant proteins with analogues found throughout nature from bacteria to higher eukaryotes. PR proteins were first noted in plants as part of the hypersensitive response, but have since been assigned an array of biological roles. The PR10/Bet v1‐like proteins are a subset of PR proteins characterized by an ability to bind a wide range of lipophilic ligands, uniquely positioning them as contributors to specialized biosynthetic pathways. PR10/Bet v1‐like proteins participate in the production of plant alkaloids and phenolics including flavonoids, both as general binding proteins and in special cases as catalysts. Owing initially to the perceived allergenic properties of PR10/Bet v1‐like proteins, many were studied at the structural level to elucidate the basis for ligand binding. These studies provided a foundation for more recent efforts to understand higher‐level structural order and how PR10/Bet v1‐like proteins catalyse key reactions in plant pathways. Synthetic biology aimed at reconstituting plant‐specialized metabolism in microorganisms uses knowledge of these proteins to fine‐tune performance in new systems.

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Section: Discussionmentioning

confidence: 99%

PR10/Bet v1‐like Proteins as Novel Contributors to Plant Biochemical Diversity

et al. 2020

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“…Operation was conducted using LTQ Tune Plus v.2.5.5 SP1 and Xcalibur v.2.1.0.1140, with additional analyses using QualBrowser feature of Xcalibur. Internal calibration, external calibration, tuning and general operations were performed using routine procedures (Morris et al ., ) optimized for alkaloid detection, with the exception that electrospray ionization (ESI), rather than heated ESI (HESI), was employed with reduced flow rates to minimize heat‐induced degradation of analytes during ionization. Data acquisition for tandem mass spectrometry was performed using a single scan event, involving CID conducted in linear ion trap (IT) with normalized collision energy (NCE) of 35%, and detection by Fourier transform mass spectrometry with resolution of 60 000 FWHM and scan range m / z 90–340.…”

Section: Methodsmentioning

confidence: 99%

“…Yeast ( S. cerevisiae strain CEN.PK) was engineered using a combination of genome integration and plasmid transformation. For genome integration, yeast was transformed by homologous recombination of integration cassettes as described previously (Morris et al ., ), which yielded strains containing T6ODM‐COR1.3 , T6ODM‐COR‐B , T6ODM‐COR1.3‐CODM and T6ODM‐COR‐B‐CODM . The dual expression pESC‐leu vector (Agilent) was used for plasmid‐based expression of T6ODM , and various COR isoforms and mutants.…”

Section: Methodsmentioning

confidence: 99%

Codeinone reductase isoforms with differential stability, efficiency and product selectivity in opium poppy

et al. 2018

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Codeinone reductase (COR) catalyzes the reversible NADPH-dependent reduction of codeinone to codeine as the penultimate step of morphine biosynthesis in opium poppy (Papaver somniferum). It also irreversibly reduces neopinone, which forms by spontaneous isomerization in aqueous solution from codeinone, to neopine. In a parallel pathway involving 3-O-demethylated analogs, COR converts morphinone to morphine, and neomorphinone to neomorphine. Similar to neopine, the formation of neomorphine by COR is irreversible. Neopine is a minor substrate for codeine O-demethylase (CODM), yielding morphine. In the plant, neopine levels are low and neomorphine has not been detected. Silencing of CODM leads to accumulation of upstream metabolites, such as codeine and thebaine, but does not result in a shift towards higher relative concentrations of neopine, suggesting a mechanism in the plant for limiting neopine production. In yeast (Saccharomyces cerevisiae) engineered to produce opiate alkaloids, the catalytic properties of COR lead to accumulation of neopine and neomorphine as major products. An isoform (COR-B) was isolated from opium poppy chemotype Bea's Choice that showed higher catalytic activity than previously characterized CORs, and it yielded mostly neopine in vitro and in engineered yeast. Five catalytically distinct COR isoforms (COR1.1-1.4 and COR-B) were used to determine sequence-function relationships that influence product selectivity. Biochemical characterization and site-directed mutagenesis of native COR isoforms identified four residues (V25, K41, F129 and W279) that affected protein stability, reaction velocity, and product selectivity and output. Improvement of COR performance coupled with an ability to guide pathway flux is necessary to facilitate commercial production of opiate alkaloids in engineered microorganisms.

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“…Genes encoding DOPA decarboxylase (DODC) from Pseudomonas putida , and norcoclaurine 6- O -methyltransferase (6OMT), coclaurine N -methyltransferase (CNMT), and 3′-hydroxy- N -methylcoclaurine 4′- O -methyltransferase 2 (4′OMT2) from Papaver somniferum were used to transform S. cerevisiae stain CENPK102-5B (MATα, ura3-52, his3D1, leu2-3/112, MAL2-8c, SUC2)40. The LiAc/PEG/single-stranded carrier DNA (ssDNA) transformation method38 was used as described previously41 To achieve plasmid-based NCS and monoamine oxidase (MAO) expression without compromising auxotrophic selection of chromosomally integrated genes, a modified version of pESC-his (Agilent) was created. An expression cassette for the KanMX4 marker was inserted immediately downstream of the HIS3 cassette to allow G418 antibiotic selection.…”

Section: Methodsmentioning

confidence: 99%

“…Relative western blot signal intensities of bands corresponding to the expected size of non-degraded NCS polypeptides were determined by digital densitometric analysis using Image Studio Lite software (LI-COR Biosciences). Liquid chromatography methods and LTQ-Orbitrap-XL mass spectrometric analysis of alkaloids was performed as described previously41.…”

Section: Methodsmentioning

confidence: 99%

Genes encoding norcoclaurine synthase occur as tandem fusions in the Papaveraceae

Lee

Chang

et al. 2016

Sci Rep

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Norcoclaurine synthase (NCS) catalyzes the enantioselective Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde as the first step in benzylisoquinoline alkaloid (BIA) biosynthesis. NCS orthologs in available transcriptome databases were screened for variants that might improve the low yield of BIAs in engineered microorganisms. Databases for 21 BIA-producing species from four plant families yielded 33 assembled contigs with homology to characterized NCS genes. Predicted translation products generated from nine contigs consisted of two to five sequential repeats, each containing most of the sequence found in single-domain enzymes. Assembled contigs containing tandem domain repeats were detected only in members of the Papaveraceae family, including opium poppy (Papaver somniferum). Fourteen cDNAs were generated from 10 species, five of which encoded NCS orthologs with repeated domains. Functional analysis of corresponding recombinant proteins yielded six active NCS enzymes, including four containing either two, three or four repeated catalytic domains. Truncation of the first 25 N-terminal amino acids from the remaining polypeptides revealed two additional enzymes. Multiple catalytic domains correlated with a proportional increase in catalytic efficiency. Expression of NCS genes in Saccharomyces cereviseae also produced active enzymes. The metabolic conversion capacity of engineered yeast positively correlated with the number of repeated domains.

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Plug-and-Play Benzylisoquinoline Alkaloid Biosynthetic Gene Discovery in Engineered Yeast

Cited by 14 publications

References 88 publications

PR10/Bet v1‐like Proteins as Novel Contributors to Plant Biochemical Diversity

PR10/Bet v1‐like Proteins as Novel Contributors to Plant Biochemical Diversity

Codeinone reductase isoforms with differential stability, efficiency and product selectivity in opium poppy

Genes encoding norcoclaurine synthase occur as tandem fusions in the Papaveraceae

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