(,Ti“cTl“.

(%ifbp wsuwʼ
hʼʼpt“ m p wʼ qp abvxʼp“b(*h ʼ(*hʼuxʼv erae ga/ ) (* ehʼp1 .,(*Glucose
NAD+NADH+H+

ʼʼx(*hP OH2ʼm4 2 ʼp
3Q 3 ʼPP
ʼ4 2ʼvʼuuxe“xnhho(*h(*h()hux H H QH   !) ,3 1 (,*/ (, ( ) (. e ( *h”
4 2! OH23 ! " !
3RKht“ mxewʼxʼʼuʼpln “xewsʼ ʼl  ʼuuxewʼ
p( )uxeym
ʼuxec &*( *hʼ ,u mxs omn “xewsov q 
7KO25 9v
7KOuuxewʼ
7KOʼ
pt(%)
uxe)%

uxe1) Sk

ʼ 5-hh1uxe
9 4hS,,%, S 1 ( )((( S 1 ( *uxePhaA (RE)BktB (RE)

O

O

R1

O

Pct (ME)
R1

OH
R2

O

R1

SCoA

NAD(P)HH+NAD(P)+HSCoA

NAD(P)HH+NAD(P)+HSCoA

Bld (CS)O

Bld (CS)

OH

OH O
OH O

R1

ME: M. elsdenii, RE: R. eutropha
CA: C. acetobutylicum, EC: E. coli
CS: C. saccharoperbutylacetonicum

V H1 %//mSCoAR2

R1 = CH3, R2 = H, Propionic acid
R1 = CH3, R2 = CH3, Isobutyric acid
R1 = OH, R2 = H, Glycolic acidOH

Hbd (CA)OH O

OH OOH

SCoA

PhaB (RE)

SCoA
R2

O

NADHH+NAD+H

NAD(P)HH+NAD(P)+R2
NAD(P)HH+NAD(P)+Adhs (EC)OHHOOH

R1 = H, R2 = H, (R)-1,3-ButanediolOH
R1 = CH3, R2 = H, (R)-1,3-Pentanediol
R
R1 = CH3, R2 = CH3, 4-Methyl-(R)-1,3-pentanediol 1
R1 = OH, R2 = H, (R)-1,2,4-Butanetriol
R2OH

100

10Growth7550125Propionate
0.100122436Time (h)4321048125
Glc

Growth

100

1075
50

4-Methyl1,3-pentanediol

125

Isobutyrate

0.100122436
Time (h)1001000
800600400
200

4-Methyl-1,3-pentanediol(μM)
Growth (OD600)

Glc

100

5Glucose, Isobutyrate (mM)

1,3-Pentanediol

125

1,3-Pentanediol (mM)
Growth (OD600)

Growth (OD600)

100

Glucose, Propionate (mM)

1,3-Diols
Glc50

Growth1040

Glycolate30
1,2,4Butanetriol

120100.1

0480122436Time (h)Glucose, Propionate
Glucose, Isobutyrate
Glucose, Glycolate

6.7 ± 0.1
6.9 ± 0.1
7.4 ± 0.1

Alcohols
1-Propanol
Isobutanol
5.84 ± 0.38
12.8 ± 0.2-40(3020102Acetyl-CoA
Acetoacetyl-CoA

pNK4

bld

phaA

hbd

8.61 ± 0.54 

20.9 ± 7.5
10.3 ± 4.80.556 ± 0.144

16.0 ± 5.510.5 ± 2.5
0.186 ± 0.057

11.1 ± 0.9
15.2 ± 1.9
14.9 ± 0.8

Not detected

U: the amount of enzyme that converts 1 µmol of the substrate to the product per minf”

t7 ?5iF 9 2A2(te )End pH(-)

1,3-BD(mM)

3-HB(mM)

Acetate
(mM)

EtOH
(mM)

α-KG
(mM)

Pyruvate
(mM)

19.3

6.1

2.90

3.16

227

29.8

14.2

1.11



(Ttft

50

NAD(P)HH+NAD(P)+h dcra(2040i40cmΔpdhR

3Formate

NADH

1Glycolytic
pathway

01020 30Time (h)40fGlcSynthetic
pathway

Acetyl-CoAYFormate

50FdhorCO2

1,3-Butanediol
NAD(P)H-type Fdh

100100NAD(P)HFdhD195Q/Y196R/Q197N

120

150

1,3-BD

NAD(P)+

Glucose

200

CO2

Fdh

280
60
NADH-type Fdh

40200() i

NAD+

250

f

20 30
Time (h)4Adhs (EC)

:03 6

Y

4CG. :036 n-,:.
4CG3 :036 :03 6t

5 10 15 20 25 30
Time (h)10Formate dehydrogenase (Fdh)

ΔpdhRfkte

500T

Parent00.5c //

40

5Growth50

10 20 30
Time (h)6(R)-1,3-BDk60

07(R)-3-Hydroxybutyraldehyde

dencg sGrowth(OD600)h

1,3-Butanediol

100TBld (CS)

NAD(P)+Acetate30.5 ± 1.7 ADPATP800PhaB (RE)

NAD(P)HH+

1JCt
rcY
fk

O2
H 2O

m100

Y

(R)-3-Hydroxybutyryl-CoA

α-Ketoglutarate(α-KG)

1,3-BD(mM)

PhaA (RE)

CoASH

48

i150

(2Formate

03-Hydroxybutyrate(3-HB)

Specific activity (U/mg Protein)
PhaA
HbdY

NADPHH+NADP+T 3F),((* 7V
2(RH(11  r2PyruvateGrowth (OD600)

TCAcycle

NAD+NADH+H+200

40Glycolyticpathway

aData are expressed as means  SD from at least three independent experiments.

7710 20 30Time (h)GlucoseMetabolites (mM)

End pH(-)

1,3-BD

0777 HH 4CG

End pH and end product data for E. coli BW25113 F’ harboring pNK8 after 48 h of fermentationa
Substrates

5Y

500Acetate

Glucose250ParentOH

60NADT

ΔpdhR0T
V H1 *%/*
m8R 2p(S)-1,3-BD

NDH-II

Acetyl-CoA NADH+H++( d 1()(GrowthO

Adhs (EC)SNNNNNN
SNNS

0.5uV H1 %/,mH

NDH-I

2QJPQND B LCHPH LO AN DN DLP N. ( 13F QPTN R SL (2NP H P T,VS
2L H P T(%SV9 T R-% I )H 9) 94R: D8( d5- 1 %. (%DLSVL H L*. 42QJPQND B LCHPH LO 4JAOI. ( 3H JO
E R SLS * S MRHYQ P O IHMMRLYB H YVLLK(, VS: D8(d5- 1 %* %DLSVL H L* 4hYO

SCoA

NADPHH+NADP+CG50R2HSCoAOOR1SCoA)))-%/*%,)(% 0*( %.
(((*777 H”gGrowth (OD600)

Glycolyticpathway

tPDHc

f

1,2,4-Butanetriol (μM)

GlucoseT

Glucose, Glycolate (mM)

(DCHQ

hh

*%
7 R

NAD+NADH+H+

1,3-BD (mM)

: D8

DLPAPH L

8RYLH)99)
H4R
9 4R
9 )C
NC f.9)4H4R)f)9)
GLHY L H
TH HRHP LYW L
96 6C
9 V9 .%( * 5P RYuxe

Pyruvate

1,3-BD (mM)h4DN

Glucose (mM)kPdhR

f

Glucose (mM)k 3F),((* 7 V / 2(RH (11     3F),((* 7  A V * 2(RH (11  3F),((* c 11 S 7 V *3F),((* c 11 S 7 V * V D* 2(RH (11S3F),((* c 11 S 7 V * V D2(RH(11Glycolyticpathway

ADPATP(*hm! Dissolved oxygen (ppm)ft“hʼʼ
H H QH  ʼuxh(*iʼmxe

00T

5 10 15 20 25 30
Time (h)Y0

5 10 15 20 25 30
Time (h)iic%k(uxv sʼTʼt”gyʼuxe(*p ʼlm eiehcyafb(*(*hhhk CGioiog4CGt(Ttcuxn( . S ʼ( *S R NRYLuxe 25 9ʼ
(,/12.*40)(,'3$+-,#c:03 6k“hh% /) S R%+ʼ