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Literature summary extracted from

  • Galanie, S.; Smolke, C.
    Optimization of yeast-based production of medicinal protoberberine alkaloids (2015), Microb. Cell Fact., 14, 144 .
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
1.5.1.31 synthesis heterologous production of berberine and the optimization of the engineered biosynthetic pathway from rac-norlaudanosoline to (S)-canadine in yeast involving the recombinant berberine reductase Berberis wilsoniae
1.6.2.4 medicine a Saccharomyces cerevisiae strain is engineered to express seven heterologous enzymes (Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR)), resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. A number of strategies are implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization. This leads to an over 70-fold increase in canadine titer up to 1.8 mg/l. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines in a microbial host. This strain is viable at pilot scale Arabidopsis thaliana
1.14.19.68 medicine a Saccharomyces cerevisiae strain is engineered to express seven heterologous enzymes (Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR)), resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. A number of strategies are implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization. This leads to an over 70-fold increase in canadine titer up to 1.8 mg/l. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines in a microbial host. This strain is viable at pilot scale Thalictrum flavum subsp. glaucum
1.21.3.3 medicine a Saccharomyces cerevisiae strain is engineered to express seven heterologous enzymes (Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR)), resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. A number of strategies are implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization. This leads to an over 70-fold increase in canadine titer up to 1.8 mg/l. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines in a microbial host. This strain is viable at pilot scale Papaver somniferum
1.21.3.3 synthesis heterologous production of berberine and the optimization of the engineered biosynthetic pathway from rac-norlaudanosoline to (S)-canadine in yeast involving the recombinant berberine reductase Papaver somniferum
2.1.1.116 medicine a Saccharomyces cerevisiae strain is engineered to express seven heterologous enzymes (Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR)), resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. A number of strategies are implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization. This leads to an over 70-fold increase in canadine titer up to 1.8 mg/l. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines in a microbial host. This strain is viable at pilot scale Papaver somniferum
2.1.1.117 medicine a Saccharomyces cerevisiae strain is engineered to express seven heterologous enzymes (Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR)), resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. A number of strategies are implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization. This leads to an over 70-fold increase in canadine titer up to 1.8 mg/l. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines in a microbial host. This strain is viable at pilot scale Thalictrum flavum subsp. glaucum
2.1.1.128 medicine a Saccharomyces cerevisiae strain is engineered to express seven heterologous enzymes (Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR)), resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. A number of strategies are implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization. This leads to an over 70-fold increase in canadine titer up to 1.8 mg/l. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines in a microbial host. This strain is viable at pilot scale Papaver somniferum
2.1.1.140 medicine a Saccharomyces cerevisiae strain is engineered to express seven heterologous enzymes (Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR)), resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. A number of strategies are implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization. This leads to an over 70-fold increase in canadine titer up to 1.8 mg/l. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines in a microbial host. This strain is viable at pilot scale Papaver somniferum

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.5.1.31 codon-optimized gene yBwSTOX is expressed in Saccharomyces cerevisiae from plasmid pCS3203, coexpression with 6 other enzyme required for production of protoberberine Berberis wilsoniae
1.6.2.4 canadine-producing Saccharomyces cerevisiae strain harbors expression cassettes for seven heterologous enzymes: Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR). The expression cassettes for the methyltransferases Ps6OMT, PsCNMT, and Ps4'OMT and the cytochrome P450 reductase CPR were chromosomally integrated, TfS9OMT and TfCAS are expressed from a high-copy plasmid, and PsBBE is expressed from a second high-copy plasmid Arabidopsis thaliana
1.14.19.68 canadine-producing Saccharomyces cerevisiae strain harbors expression cassettes for seven heterologous enzymes: Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR). The expression cassettes for the methyltransferases Ps6OMT, PsCNMT, and Ps4'OMT and the cytochrome P450 reductase CPR were chromosomally integrated, TfS9OMT and TfCAS are expressed from a high-copy plasmid, and PsBBE is expressed from a second high-copy plasmid Thalictrum flavum subsp. glaucum
1.21.3.3 canadine-producing Saccharomyces cerevisiae strain harbors expression cassettes for seven heterologous enzymes: Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR). The expression cassettes for the methyltransferases Ps6OMT, PsCNMT, and Ps4'OMT and the cytochrome P450 reductase CPR were chromosomally integrated, TfS9OMT and TfCAS are expressed from a high-copy plasmid, and PsBBE is expressed from a second high-copy plasmid Papaver somniferum
2.1.1.116 canadine-producing Saccharomyces cerevisiae strain harbors expression cassettes for seven heterologous enzymes: Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR). The expression cassettes for the methyltransferases Ps6OMT, PsCNMT, and Ps4'OMT and the cytochrome P450 reductase CPR were chromosomally integrated, TfS9OMT and TfCAS are expressed from a high-copy plasmid, and PsBBE is expressed from a second high-copy plasmid Papaver somniferum
2.1.1.117 canadine-producing Saccharomyces cerevisiae strain harbors expression cassettes for seven heterologous enzymes: Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR). The expression cassettes for the methyltransferases Ps6OMT, PsCNMT, and Ps4'OMT and the cytochrome P450 reductase CPR were chromosomally integrated, TfS9OMT and TfCAS are expressed from a high-copy plasmid, and PsBBE is expressed from a second high-copy plasmid Thalictrum flavum subsp. glaucum
2.1.1.128 canadine-producing Saccharomyces cerevisiae strain harbors expression cassettes for seven heterologous enzymes: Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR). The expression cassettes for the methyltransferases Ps6OMT, PsCNMT, and Ps4'OMT and the cytochrome P450 reductase CPR were chromosomally integrated, TfS9OMT and TfCAS are expressed from a high-copy plasmid, and PsBBE is expressed from a second high-copy plasmid Papaver somniferum
2.1.1.140 canadine-producing Saccharomyces cerevisiae strain harbors expression cassettes for seven heterologous enzymes: Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR). The expression cassettes for the methyltransferases Ps6OMT, PsCNMT, and Ps4'OMT and the cytochrome P450 reductase CPR were chromosomally integrated, TfS9OMT and TfCAS are expressed from a high-copy plasmid, and PsBBE is expressed from a second high-copy plasmid Papaver somniferum

Protein Variants

EC Number Protein Variants Comment Organism
1.5.1.31 additional information recombinant coexpression of enzyme STOX and six other enzymes, including the three membrane-bound enzymes: the flavin-dependent oxidase berberine bridge enzyme, the cytochrome P450 canadine synthase, and a cytochrome P450 reductase, in Saccharomyces cerevisiae results in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor rac-norlaudanosoline. Improvement of flux through the pathway, by methods including enzyme variant screening, genetic copy number variation, and culture optimization, leading to an over 70fold increase in canadine titer up to 1.8 mg/l Berberis wilsoniae
1.21.3.3 additional information recombinant coexpression of seven enzymes, including the three membrane-bound enzymes: the flavin-dependent oxidase berberine bridge enzyme, the cytochrome P450 canadine synthase, and a cytochrome P450 reductase, in Saccharomyces cerevisiae results in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor rac-norlaudanosoline. Improvement of flux through the pathway, by methods including enzyme variant screening, genetic copy number variation, and culture optimization, leading to an over 70fold increase in canadine titer up to 1.8 mg/l Papaver somniferum

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
1.14.19.68 membrane bound to Thalictrum flavum subsp. glaucum 16020
-
1.21.3.3 membrane membrane-bound Papaver somniferum 16020
-

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.5.1.31 (R)-canadine + 2 NADP+ Berberis wilsoniae
-
 berberine + 2 NADPH + H+
-
 ?
1.21.3.3 (S)-reticuline + O2 Papaver somniferum
-
 (S)-scoulerine + H2O2
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.31 Berberis wilsoniae
-
-
-
1.6.2.4 Arabidopsis thaliana Q9SB48
-
-
1.14.19.68 Thalictrum flavum subsp. glaucum Q5C9L1
-
-
1.21.3.3 Papaver somniferum P93479
-
-
2.1.1.116 Papaver somniferum Q7XB10
-
-
2.1.1.117 Thalictrum flavum subsp. glaucum Q5C9L2
-
-
2.1.1.128 Papaver somniferum Q6WUC1
-
-
2.1.1.140 Papaver somniferum Q7XB08
-
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.31 (R)-canadine + 2 NADP+
-
 Berberis wilsoniae berberine + 2 NADPH + H+
-
 ?
1.21.3.3 (S)-reticuline + O2
-
 Papaver somniferum (S)-scoulerine + H2O2
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.5.1.31 (S)-tetrahydroprotoberberine oxidase
-
 Berberis wilsoniae
1.5.1.31 STOX
-
 Berberis wilsoniae
1.21.3.3 BBE
-
 Papaver somniferum
1.21.3.3 BBE1
-
 Papaver somniferum
1.21.3.3 berberine bridge enzyme
-
 Papaver somniferum
1.21.3.3 flavin-dependent oxidase
-
 Papaver somniferum
1.21.3.3 reticuline oxidase
-
 Papaver somniferum

Cofactor

EC Number Cofactor Comment Organism Structure
1.5.1.31 NADP+
-
 Berberis wilsoniae
1.21.3.3 FAD
-
 Papaver somniferum