Nature - USA (2020-09-24)

Nature | Vol 585 | 24 September 2020 | 615

precursors in yeast (Fig. 1a). Modules I/II and III enable de novo biosyn-
thesis of the acyl acceptor and donor moieties; module IV enables TA
scaffold modifications to produce hyoscyamine and scopolamine; mod-
ule V comprises the central acyltransferase reaction linking upstream
acyl acceptor/donor biosynthesis to downstream scaffold modifica-
tions. As a starting point, we used a yeast platform strain (CSY1251) that
was previously engineered for de novo production of the acyl acceptor

tropine via modules I and II^14 (Extended Data Fig. 1). A putrescine bio-

synthesis module (I) designed to increase putrescine accumulation

incorporated (i) overexpression of glutamate N-acetyltransferase

(Arg2), arginase (Car1), ornithine decarboxylase (Spe1) and polyamine

oxidase (Fms1); (ii) a parallel plant/bacterial pathway encoded by Avena

sativa arginine decarboxylase (AsADC) and Escherichia coli agmatine

ureohydrolase (speB); and (iii) disruptions to polyamine regulatory

Vacuole

TA scafModule V:fold biosynthesis^ -AbLSDsRed

NtJAT1

AsADC

Fms1

Fms1

Spe1

Car1

speB

Arginine Glutamic acid

Agmatine Ornithine

Putrescine

Spermine

Spermidine

Meu1

Oaz1

Mitochondrion

PutrescineModuleI:

biosynthesis

Arg2

Aro8Aro9

Egh1

Phenylalanine

Phenylpyruvic acid

Phenyllactic acidglucoside Phenyllactic acid

WfPPR

AbUGT

PLAModule III: glucoside

biosynthesis

Tropine biosynthesisModule II:

Ald2 Hfd1Ald3

Ald4 Ald5 4-methylaminobutanal

pyrroliniumN-methyl-

4-(1-Methyl-2-pyrrodinyl)-

3-oxobutanoic acid

Tropinone Tropine

N-methylputrescine DmMPO1ΔC-PTS1

DsTR1

AbDsPMT1PMT1

Spontaneou

s

AbAtCYP82M3ATR1

2× malonylAbPYKS

-CoA

Peroxisome

AbAtCYP80F1ATR1

Hyoscyaminealdehyde

Hyoscyamine

Scopolamine

Littorine

unknownPreviously

DsHDH

DsH6H

Medicinal TAModule IV biosynthesis:

Nucleus ER

HO

O

NH 2

OH

O

H 2 N NH

NH

NH 2

OH

O

H 2 N N

NH 2

NH 2 H 2 N

NH 2

OH

O

H 2 N NH^2

H 2 N NH

HN NH 2

H 2 N

HN NH 2

H 2 N

HN

H 2 N

HN

O

HN

O

HN

N

N

O

HO

O

N

O

N

OH

NH 2

OH

O

O

OH

O

OH

OH

O

OH

OGlc

O

N

O

O

N OH

O

O

OH

N

O

O

O

N

O

O

OH

N

O

O

OH

O

OH

OGlc

N O

OH

a

d

Relative PLA

glucoside titre

ControlUGP1PGM2

0.0

0.5

1.0

1.5

2.0 *

e

Relative PLA glucoside titre

ControlΔEXG1ΔSPR1ΔEGH1

0.0

0.5

1.0

1.5

2.0

2.5

*

***

c PLAPLA

Tropine glucoside

1251

1287

1288

4567

Time (min)

3456

Time (min)

NA

0123

Time (min)

Stds

b CSY#Ļ

PLA titre (mg l

–1)

ControlBcLDHLcLD

H

LpLD

H

LpPP

R

hcx

B

AbPP

R

WfPPR

0

5

10

(^15015)
200
250
300

• 
  
  
  
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  Fig. 1 | Engineered biosynthetic pathway for de novo production of scopolamine
  in yeast and optimization of PLA-glucoside biosynthesis. a, Modular pathway
  construction for scopolamine biosynthesis in yeast. Enzyme/protein colour scheme:
  orange, yeast (overexpressed); green, plant; purple, bacteria; red, other eukaryote;
  grey, spontaneous/non-enzymatic. Red boxes indicate disrupted yeast proteins;
  dotted or solid lines of vacuole membrane delineate functional biosynthetic
  modules. DsRed–AbLS, Discosoma sp. red fluorescent protein fused to the N
  terminus of A. belladonna littorine synthase. b, PLA production in yeast engineered
  for expression of PPRs or LDHs. Heterologous enzymes or negative control (BFP)
  were expressed from low-copy plasmids in strain CSY1251. c, Multiple reaction
  monitoring (MRM) and extracted ion chromatogram (EIC) traces from culture
  medium of yeast engineered for step-wise reconstitution of PLA glucoside
  biosynthesis via module III. Chromatogram traces are representative of three
  biological replicates. d, Relative titres of PLA glucoside in yeast engineered for
  overexpression of UDP-glucose biosynthetic enzymes. Enzymes or negative control
  (BFP) were expressed from low-copy plasmids in strain CSY1288. e, Relative PLA
  glucoside titres in CSY1288 with disruptions to endogenous glucosidases. In d and e,
  PLA glucoside accumulation was compared using relative titres owing to lack of an
  authentic chemical standard. Strains were cultured for 72 h before liquid
  chromatography–tandem mass spectrometry (LC–MS/MS) analysis of metabolites
  in culture supernatant. Data in b, d and e represent the mean of n = 3 biologically
  independent samples (open circles), error bars denote s.d. *P < 0.05, *P < 0.01,
  P < 0.001, Student’s two-tailed t-test. Statistical significance is shown relative to
  controls. Exact P values are in Supplementary Table 5.