1.1.1.415

synthesis

complete biosynthesis of noscapine and halogenated alkaloids in yeast and optimizing noscapine production toward scalable manufacturing. Engineered strain contains 25 heterologous plant, bacteria, and mammalian genes and 6 mutant or overexpressed yeast genes. The noscapine biosynthetic pathway incorporates seven endomembrane-localized plant enzymes, highlighting the ability of the yeast to functionally express and properly localize large numbers of heterologous enzymes into the endoplasm reticulum. Noscapine titers were improved by 18000fold (to low mg/l levels) via a combination of enzyme engineering, pathway and strain engineering, and fermentation optimization. Microbial fermentation can be used to produce halogenated alkaloid derivatives, which can ultimately serve as potential drug leads, through feeding amino acid derivatives to strains

Papaver somniferum

1.14.14.163

synthesis

de novo production of noscapine in Saccharomyces cerevisiae, through the reconstruction of a biosynthetic pathway comprising over 30 enzymes from plants, bacteria, mammals, and yeast itself, including 7 plant endoplasmic reticulum (ER)-localized enzymes. Optimization directed to tuning expression of pathway enzymes, host endogenous metabolic pathways, and fermentation conditions led to an over 18,000-fold improvement from initial noscapine titers to 2.2 mg/l

Papaver somniferum

2.3.1.285

synthesis

de novo production of noscapine in Saccharomyces cerevisiae, through the reconstruction of a biosynthetic pathway comprising over 30 enzymes from plants, bacteria, mammals, and yeast itself. Optimization of conditions leads to an over 18,000fold improvement from initial noscapine titers to about2.2 mg/l. By feeding modified tyrosine derivatives to the optimized noscapine-producing strain, microbial production of halogenated benzylisoquinoline alkaloids is possible

Papaver somniferum

1.1.1.415

Saccharomyces cerevisiae CSY1060 and CSY1061 strains are used as the base strain for construction of the de novo noscapine biosynthetic strains

Papaver somniferum

1.14.14.166

expression in Saccharomyces cerevisiae

Papaver somniferum

1.1.1.415

narcotine hemiacetal + NAD+

Papaver somniferum

noscapine + NADH + H+

?

1.14.14.163

(S)-1-hydroxy-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

Papaver somniferum

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine

(13S,14R)-1,13-dihydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.14.14.166

(S)-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

Papaver somniferum

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine. The pathway for biosynthesis of noscapine, a potential anticancer compound, from canadine is a 10-gene cluster

(S)-1-hydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.14.14.167

(13S,14R)-13-O-acetyl-1-hydroxy-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

Papaver somniferum

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine

(13S,14R)-13-O-acetyl-1,8-dihydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.1.1.415

Papaver somniferum

I3PLR3

1.14.14.163

Papaver somniferum

I3PLR0

1.14.14.166

Papaver somniferum

I3PLR1

1.14.14.167

Papaver somniferum

I3V6B7

2.3.1.285

Papaver somniferum

I3PLR4

1.1.1.415

narcotine hemiacetal + NAD+

749191

Papaver somniferum

noscapine + NADH + H+

?

1.14.14.163

(S)-1-hydroxy-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

749191

Papaver somniferum

(13S,14R)-1,13-dihydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.14.14.163

(S)-1-hydroxy-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine

749191

Papaver somniferum

(13S,14R)-1,13-dihydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.14.14.166

(S)-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

749191

Papaver somniferum

(S)-1-hydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.14.14.166

(S)-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine. The pathway for biosynthesis of noscapine, a potential anticancer compound, from canadine is a 10-gene cluster

749191

Papaver somniferum

(S)-1-hydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.14.14.167

(13S,14R)-13-O-acetyl-1-hydroxy-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

749191

Papaver somniferum

(13S,14R)-13-O-acetyl-1,8-dihydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.14.14.167

(13S,14R)-13-O-acetyl-1-hydroxy-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine

749191

Papaver somniferum

(13S,14R)-13-O-acetyl-1,8-dihydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

2.3.1.285

acetyl-CoA + (13S,14R)-1,13-dihydroxy-N-methylcanadine

749191

Papaver somniferum

(13S,14R)-13-O-acetyl-1-hydroxy-N-methylcanadine + CoA

?

1.1.1.415

SDR1

Papaver somniferum

1.1.1.415

short-chain dehydrogenase/reductase

Papaver somniferum

1.14.14.163

1-hydroxy-N-methylcanadine 13-hydroxylase

Papaver somniferum

1.14.14.163

CYP82X2

Papaver somniferum

1.14.14.166

1-hydroxy-N-methylcanadine synthase

Papaver somniferum

1.14.14.166

CYP82Y1

Papaver somniferum

1.14.14.167

1-hydroxy-13-O-acetyl-N-methylcanadine 8-hydroxylase

Papaver somniferum

1.14.14.167

CYP82X1

Papaver somniferum

1.14.14.166

cytochrome P-450

heme-thiolate enzyme

Papaver somniferum

1.14.14.167

cytochrome P-450

heme-thiolate enzyme

Papaver somniferum

1.1.1.415

synthesis

complete biosynthesis of noscapine and halogenated alkaloids in yeast and optimizing noscapine production toward scalable manufacturing. Engineered strain contains 25 heterologous plant, bacteria, and mammalian genes and 6 mutant or overexpressed yeast genes. The noscapine biosynthetic pathway incorporates seven endomembrane-localized plant enzymes, highlighting the ability of the yeast to functionally express and properly localize large numbers of heterologous enzymes into the endoplasm reticulum. Noscapine titers were improved by 18000fold (to low mg/l levels) via a combination of enzyme engineering, pathway and strain engineering, and fermentation optimization. Microbial fermentation can be used to produce halogenated alkaloid derivatives, which can ultimately serve as potential drug leads, through feeding amino acid derivatives to strains

Papaver somniferum

1.14.14.163

synthesis

de novo production of noscapine in Saccharomyces cerevisiae, through the reconstruction of a biosynthetic pathway comprising over 30 enzymes from plants, bacteria, mammals, and yeast itself, including 7 plant endoplasmic reticulum (ER)-localized enzymes. Optimization directed to tuning expression of pathway enzymes, host endogenous metabolic pathways, and fermentation conditions led to an over 18,000-fold improvement from initial noscapine titers to 2.2 mg/l

Papaver somniferum

2.3.1.285

synthesis

de novo production of noscapine in Saccharomyces cerevisiae, through the reconstruction of a biosynthetic pathway comprising over 30 enzymes from plants, bacteria, mammals, and yeast itself. Optimization of conditions leads to an over 18,000fold improvement from initial noscapine titers to about2.2 mg/l. By feeding modified tyrosine derivatives to the optimized noscapine-producing strain, microbial production of halogenated benzylisoquinoline alkaloids is possible

Papaver somniferum

1.1.1.415

Saccharomyces cerevisiae CSY1060 and CSY1061 strains are used as the base strain for construction of the de novo noscapine biosynthetic strains

Papaver somniferum

1.14.14.166

expression in Saccharomyces cerevisiae

Papaver somniferum

1.14.14.166

cytochrome P-450

heme-thiolate enzyme

Papaver somniferum

1.14.14.167

cytochrome P-450

heme-thiolate enzyme

Papaver somniferum

1.1.1.415

narcotine hemiacetal + NAD+

Papaver somniferum

noscapine + NADH + H+

?

1.14.14.163

(S)-1-hydroxy-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

Papaver somniferum

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine

(13S,14R)-1,13-dihydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.14.14.166

(S)-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

Papaver somniferum

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine. The pathway for biosynthesis of noscapine, a potential anticancer compound, from canadine is a 10-gene cluster

(S)-1-hydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.14.14.167

(13S,14R)-13-O-acetyl-1-hydroxy-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

Papaver somniferum

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine

(13S,14R)-13-O-acetyl-1,8-dihydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.1.1.415

narcotine hemiacetal + NAD+

749191

Papaver somniferum

noscapine + NADH + H+

?

1.14.14.163

(S)-1-hydroxy-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

749191

Papaver somniferum

(13S,14R)-1,13-dihydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.14.14.163

(S)-1-hydroxy-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine

749191

Papaver somniferum

(13S,14R)-1,13-dihydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.14.14.166

(S)-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

749191

Papaver somniferum

(S)-1-hydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.14.14.166

(S)-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine. The pathway for biosynthesis of noscapine, a potential anticancer compound, from canadine is a 10-gene cluster

749191

Papaver somniferum

(S)-1-hydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.14.14.167

(13S,14R)-13-O-acetyl-1-hydroxy-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

749191

Papaver somniferum

(13S,14R)-13-O-acetyl-1,8-dihydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

1.14.14.167

(13S,14R)-13-O-acetyl-1-hydroxy-N-methylcanadine + [reduced NADPH-hemoprotein reductase] + O2

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine

749191

Papaver somniferum

(13S,14R)-13-O-acetyl-1,8-dihydroxy-N-methylcanadine + [oxidized NADPH-hemoprotein reductase] + H2O

?

2.3.1.285

acetyl-CoA + (13S,14R)-1,13-dihydroxy-N-methylcanadine

749191

Papaver somniferum

(13S,14R)-13-O-acetyl-1-hydroxy-N-methylcanadine + CoA

?

1.14.14.163

metabolism

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine

Papaver somniferum

1.14.14.166

metabolism

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine

Papaver somniferum

1.14.14.167

metabolism

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine

Papaver somniferum

1.14.14.163

metabolism

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine

Papaver somniferum

1.14.14.166

metabolism

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine

Papaver somniferum

1.14.14.167

metabolism

the enzyme participates in the biosynthesis of the isoquinoline alkaloid noscapine

Papaver somniferum