Time (hr)

0 12 24 36 48 60

Xylo

se (

g L

-1)

0

10

20

30

40

A6

00, X

ylit

ol, G

lycero

l, a

nd E

thanol (g

L-1

)

0

2

4

6

8

10

12

Xylose Xylitol Glycerol

Ethanol A600

Supplementary Figure S1. Anaerobic xylose fermentation profile by recombinant S.

cerevisiae strain SR8. Results are the mean of duplicate experiments; error bars represent

standard deviations and are not visible when smaller than the symbol size.

Acetoin amendment (g L-1

)

5 10 15 22 30

Consu

mption o

r pro

duction o

f com

pounds (g L

-1)

0

2

4

6

8

10

12

14

Acetoin consumption

2,3-butanediol production

a

Acetoin amendment (g L-1

)

no acetoin 5 10 15 22 30

Eth

an

ol yie

ld (

g p

er

g s

uga

r)

0.30

0.32

0.34

0.36

0.38

0.40

0.42

Gly

ce

rol a

nd

xylit

ol yie

ld (

g p

er

g s

uga

r)

0.00

0.05

0.10

0.15

0.20

0.25ethanol yield

glycerol yield

xylitol yield

b

X: Decrease of glycerol and xlylitol accumulation (mmole)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

Y: N

AD

+ r

eg

en

era

te b

y a

ce

toin

re

du

ctio

n (

mm

ole

)

0.0

0.5

1.0

1.5

2.0

Y= 1.37*X, R2= 0.983

c

Supplementary Figure S2. Quantification of the consumption of acetoin and production

of 2R,3R-butanediol in xylose fermentation experiments with different acetoin

amendments (a) and evaluation of xylose fermentation improvement in terms of yield of

ethanol, glycerol and xylitol by using acetoin as an external electron sink at different

concentrations(b). The following reaction can occur in S. cerevisiae: acetoin + NADH

2R,3R-butanediol + NAD+ (EC 1.1.1.4). Results are the mean of duplicate experiments

and error bars indicate standard deviations. (c) Correlation between NAD+ regenerated

by external electron acceptor acetoin reduction and the decrease in glycerol and xylitol

accumulation during anaerobic xylose fermentation by S. cerevisae SR8. The grey

straight line represents the linear regression curve; fitting equation and R2 are given.

Supplementary Figure S3. EFM analysis of the acetate reduction pathway in yeast. The

box-and-whisker plots show a comparison between the xylose only consuming EFMs

(blue) and the xylose and acetate consuming EFMs (green) for both ethanol yield (a) and

glycerol yield (b). The median (thick red central line) is shown with the 25th

and 75th

percentile ranges (box depth) and the maximum and minimum values (T-bars).

0.1

0.2

0.3

0.4

0.5

0.6

xylose-only consumingmodes

xylose and acetate co-consuming modes

Eth

ano

l Yie

ld (

g p

er

g xy

lose

)

0

0.1

0.2

0.3

0.4

0.5

0.6

xylose-only consumingmodes

xylose and acetate co-consuming modes

Gly

cero

l Yie

ld (

g p

er

g xy

lose

)

b

a

S-adhE S-eutE S-PadhE S-nc

En

zym

atic a

ctivity (

mm

ole

g p

rote

in-1

min

-1)

0

1

2

3

4

5

6

Supplementary Figure. S4. Enzymatic activity assays of NADH-dependent acetylating

acetaldehyde dehydrogenase reaction in the three yeast strains active in anaerobic acetate

assimilation compared to the control strain. Results are the mean of duplicate

experiments and error bars indicate standard deviations.

a

b

Supplementary Figure. S5. Extracted chromatographs of ethanol peak for specific ion

fragments 47, 46, 45, 32, and 31 from GC-MS analysis of the samples from S-adhE

fermentation (a) and S-nc fermentation (b). A representative experiment is shown from

one of two independent replicates, which differed by less than 5%.

1 . 7 2 1 . 7 4 1 . 7 6 1 . 7 8 1 . 8 0 1 . 8 2 1 . 8 4 1 . 8 6 1 . 8 8 1 . 9 0 1 . 9 2 1 . 9 4 1 . 9 6 1 . 9 80

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

8 0 0 0 0

1 0 0 0 0 0

1 2 0 0 0 0

1 4 0 0 0 0

1 6 0 0 0 0

1 8 0 0 0 0

2 0 0 0 0 0

2 2 0 0 0 0

2 4 0 0 0 0

2 6 0 0 0 0

2 8 0 0 0 0

3 0 0 0 0 0

3 2 0 0 0 0

3 4 0 0 0 0

3 6 0 0 0 0

3 8 0 0 0 0

4 0 0 0 0 0

T i m e - - >

A b u n d a n c e

I o n 4 7 . 0 0 ( 4 6 . 7 0 t o 4 7 . 7 0 ) : S a d h E . D \ d a t a . m sI o n 4 6 . 0 0 ( 4 5 . 7 0 t o 4 6 . 7 0 ) : S a d h E . D \ d a t a . m sI o n 4 5 . 0 0 ( 4 4 . 7 0 t o 4 5 . 7 0 ) : S a d h E . D \ d a t a . m sI o n 3 2 . 0 0 ( 3 1 . 7 0 t o 3 2 . 7 0 ) : S a d h E . D \ d a t a . m sI o n 3 1 . 0 0 ( 3 0 . 7 0 t o 3 1 . 7 0 ) : S a d h E . D \ d a t a . m s

1 . 7 2 1 . 7 4 1 . 7 6 1 . 7 8 1 . 8 0 1 . 8 2 1 . 8 4 1 . 8 6 1 . 8 8 1 . 9 0 1 . 9 2 1 . 9 4 1 . 9 6 1 . 9 80

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

4 0 0 0 0

5 0 0 0 0

6 0 0 0 0

7 0 0 0 0

8 0 0 0 0

9 0 0 0 0

1 0 0 0 0 0

1 1 0 0 0 0

1 2 0 0 0 0

1 3 0 0 0 0

1 4 0 0 0 0

1 5 0 0 0 0

1 6 0 0 0 0

1 7 0 0 0 0

T im e - ->

A b u n d a n c e

I o n 4 7 . 0 0 (4 6 . 7 0 t o 4 7 . 7 0 ) : S n c t 2 -3 . D \ d a t a . m sI o n 4 6 . 0 0 (4 5 . 7 0 t o 4 6 . 7 0 ) : S n c t 2 -3 . D \ d a t a . m sI o n 4 5 . 0 0 (4 4 . 7 0 t o 4 5 . 7 0 ) : S n c t 2 -3 . D \ d a t a . m sI o n 3 2 . 0 0 (3 1 . 7 0 t o 3 2 . 7 0 ) : S n c t 2 -3 . D \ d a t a . m sI o n 3 1 . 0 0 (3 0 . 7 0 t o 3 1 . 7 0 ) : S n c t 2 -3 . D \ d a t a . m s

a b

S-adhE S-eutE S-mphF S-PadhES-Cbaldh S-nc

Eth

ano

l yie

ld (

g p

er

g x

ylo

se)

0.30

0.32

0.34

0.36

0.38

S-adhE S-eutE S-mphFS-PadhES-Cbaldh S-nc

Specific

eth

anol pro

ductivity (

g g

bio

mass

-1 h

r- 1)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

c

S-adhE S-eutE S-mphFS-PadhES-Cbaldh S-nc

Bypro

duct yie

ld (

g p

er

g x

ylo

se

)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Supplementary Figure S6. Ethanol yields (a), specific ethanol productivity (b), and

byproduct (glycerol and xylitol) yields (c) from anaerobic xylose fermentation by

different S. cerevisiae strains. Results are the mean of duplicate experiments and error

bars indicate standard deviations.

Aceta

te c

onsum

ption (

g L

-1)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

S-adhE S-nc

a

Eth

an

ol yie

lds (

g p

er

g s

ub

str

ate

)

0.30

0.31

0.32

0.33

0.34

0.35

0.36

0.37

0.38

0.39

S-adhE S-nc

b

Time (hr)

0 20 40 60 80 100 120 140

O.D

.(6

00

nm

)

0

1

2

3

4

S-adhE

S-nc

c

Supplementary Figure S7. Cumulative acetate consumption (a), ethanol yields (b) and

cell growth (OD600) (c) comparison by S. cerevisiae strains S-adhE and the control S-nc

during anaerobic xylose fermentation in medium buffered at pH 4.7. Results are the

mean of duplicate experiments and error bars indicate standard deviations.

Ace

tate

co

nsu

mp

tio

n (

g L

-1)

-0.10

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

SR7-adhE SR7-nc

a

E

thanol yie

lds (

g p

er

g s

ubstr

ate

)

0.30

0.31

0.32

0.33

0.34

0.35

0.36

0.37

SR7-adhE SR7-nc

b

Supplementary Figure S8. Cumulative acetate consumption (a) and ethanol yields (b)

from anaerobic xylose fermentation by S. cerevisiae strains SR7-adhE and the control

SR7-nc. Results are the mean of duplicate experiments and error bars indicate standard

deviations.

S-adhE S-nc

Accu

mu

lative

aceta

te c

onsu

mp

tion

(g

L-1

)

0.0

0.2

0.4

0.6

0.8

1.0

a

S-adhE S-nc

Eth

an

ol yie

ld (

g p

er

g s

ubstr

ate

)0.30

0.32

0.34

0.36

0.38

b

S-adhE S-nc

Ma

xim

um

sp

ecific

gro

wth

ra

te (

hr-1

)

0.000

0.005

0.010

0.015

0.020

0.025

c

Supplementary Figure S9. Cumulative anaerobic acetate consumption (a), ethanol

yields (b), and specific growth rates (c) during fermentation by SadhE strain and Snc

strain in synthetic complete medium containing 40 g L-1

xylose, 20 g L-1

glucose and 2 g

L-1

acetate. Results are the mean of duplicate experiments and error bars indicate

standard deviations.

S-adhE (i) S-nc (i) S-adhE (ii) S-nc (ii)

Accum

ula

tive a

ceta

te c

onsum

ption (

g L

-1)

0.0

0.1

0.2

0.3

0.4

a

S-adhE (i) S-nc (i) S-adhE (ii) S-nc (ii)

Eth

anol yie

ld (

g p

er

g s

ug

ar)

0.30

0.32

0.34

0.36

0.38

0.40

0.42

b

S-adhE (i) S-nc (i)

Ma

xim

um

sp

ecific

gro

wth

ra

te (

hr-1

)

0.00

0.01

0.02

0.03

c

Supplementary Figure S10. Cumulative anaerobic acetate consumption (a), ethanol

yields (b), and specific growth rates (c) during fermentation of corn stover hydrolysates

by SadhE strain and Snc strain (i) without the addition of cellulase and cellobiase or (ii)

with the addition of cellulase and cellobiase. Results are the mean of duplicate

experiments and error bars indicate standard deviations.

Supplementary Table S1. Summary of representative up-to-date engineered xylose-fermenting S. cerevisiae strains

Stains Characteristics Conditions rXylose rXylose* YXylitol YEthanol VEthanol PEthanol* Initial cell

density ( g

L-1

)

Refs

SR8 XYL1, XYL2,

XYL3 (two copies

for each gene),

Δald6, evolved.

Oxygen limited,

YPX (80 g L-1

)

2.1 0.74 0.05 0.37 0.79 0.28 0.3 This

study

Anaerobic, YPX

(40 g L-1

)

0.64 0.72 0.12 0.35 0.21 0.24 0.3 This

study

Anaerobic, SCX 40

g L-1

1.73 0.65 0.08 0.39 0.67 0.25 2 This

study

TMB3400 XYL1, XYL2,

XKS1, random

mutagenesis

Anaerobic, minimal

media +xylose (20

g L-1

)

- - 0.25 0.18 0.11 0.024 0.02 40

TMB

3001C1

XYL1, XYL2,

XKS1, evolved

Anaerobic, minimal

media+xylose (10

g L-1

)

- 0.60 0.32 0.28 - - - 41

TMB3271 XYL1 (K270M,

two copies), XYL2,

XKS1

Oxygen limited,

minimal media

+xylose (50 g L-1

)

0.23 - 0.09 0.31 0.28 - 5 42

DR PHO13 XYL1, XYL2,

XYL3, pho13Δ

Aerobic, YNBX(40

g L-1

)

- 0.37 0.04 0.25 - 0.093 - 43

CMB.JHV.X

YL123

pha13a

XYL1, XYL2,

XYL3, pho13Δ

Low oxygen,

defined minimal

media, xylose (20

g L-1

)

0.47 0.14 0.006 0.24 0.047 0.015 - 44

MA-R5 XYL1, mXYL2

(D207A/I208R/F2

09S/N211R), XKS

Anaerobic YPX (45

g L-1

)

- - - 0.37 0.50 0.12 2.8 45

TMB3420

(selected

population)

mXYL1

(N272D/P275Q),

XYL2, XKS1,

TAL1, TKL1,

RPE1, RKI1,

Δgre3, evolved

Anaerobic, SCX

(60 g L-1

)

- 0.89 0.13 0.36 - 0.32 - 46

DA24-16 XYL1, m XYL1,

XYL2, and XKS1,

evolved

Oxygen limited,

YPX (80 g L-1

)

- 0.71 0.04 0.35 - 0.25 0.3 47

BY4741X/Δ

PHO13

XYL1, XYL2,

XKS1, Δpho13

Oxygen-limited,

YPX (80 g L-1

)

0.435 - 0.36 2.2 0.15 - 48

SX6MUT

mXYL1, XYL2,

XKS1, TAL, ald6Δ

Oxygen limited, SC

with xylose (40 g L-

1) and glucose (20 g

L-1

)

0.64 0.06 0.037 0.39 0.25 0.02 2.5-3.3 49

TMB3102 XylA, gre3Δ Anaerobic, YNBX

( 50 g L-1

)

- - 0.31 0.21 0.004 0.0004 - 50

TMB3112 XylA, XKS1, gre3Δ Anaerobic,

synthetic media

xylose ( 50 g L-1

)

- 0.006 - 0.14 0.009 0.0009 10 51

TMB3050 XylA, XKS1,TAL1,

TKL1, RKI1,

RPE1, gre3Δ,

evolved

Oxygen limited,

synthetic media

xylose ( 50 g L-1

)

- 0.002

4

0.23 0.29 - - - 52

RWB 218 XylA, XKS1, TAL1,

TKL1, RPE1,

RKI1, gre3Δ ,

evolved

Anaerobic, SCX

(20 g L-1

)

- 1.2 - 0.41 - 0.49 0.2 53, 54

ADAP8 XylA, XKS1,

SUT1, evolved

Anaerobic, SCX

(20 g L-1

)

0.07

1

- 0.17 0.37 0.026 - - 55

Note:

rXylose: xylose consumption rate (g L-1

hr-1

)

rXylose*: specific xylose consumption rate (hr-1

)

Yxylitol: xylitol yield (g xylitol per g xylose)

Yethanol: ethanol yield (g ethanol per g xylose)

Vethanol: volumetric ethanol productivity (g L-1

hr-1

)

Pethanol*: specific ethanol productivity (hr-1

)

XYL1, xylose reductase from Scheffersomyces stipitis; XYL2, xylitol dehydrogenase from S. stipitis; XYL3, xylulokinase from

S. stipitis; XKS1, Xylulokinase from S. cerevisiae; GAPDH, glyceraldehyde 3-phosphate dehydrogenase from S. cerevisiae;

MAE1, malic enzyme from S. cerevisiae; MDH2, malate dehydrogenase from S. cerevisiae; PYC2, pyruvate carboxylase from

S. cerevisiae; pho13 alkaline phosphatase from S. cerevisiae; TAL1 transaldolase from S. cerevisiae; TKL1, transketolase from

S. cerevisiae; RPE1 ribulose-5-phosphate 3-epimerase from S. cerevisiae; RKI1 ribose-5-phosphate ketol-isomerase from S.

cerevisiae; gre3, aldose reductase from S. cerevisiae; ald6, aldehyde dehydrogenase from S. cerevisiae; cox4, cytochrome c

oxidase from S. cerevisiae; XylA, xylose isomerase.

H131-A3-

ALCS

XylA, XYL3, TAL1,

TKL1, RPE1,

RKI1, evolved

Anaerobic, SCX

(40 g L-1

)

- 1.87 <0.01 0.41 - 0.77 0.1 34

BY4741-

S2A3K

mXylA, TAL1,

XKS1, gre3Δ

Microaerobic,

YSCX (40 g L-1

).

- 0.057 - 0.42 - 0.024 3.4 56

Supplementary Table S2. Summary of product yields, acetate consumption, and NAD+

generation in anaerobic fermentation of xylose and acetate by the recombinant yeast

strains

Note: Average values of duplicate experiments are reported. Deviations between the duplicate are

less than 2%. a. ethanol yields are calculated by considering both xylose and acetate as substrates.

Supplementary Table S3. Intracellular concentration of coenzymes in the recombinant

yeast strains during anaerobic xylose or glucose fermentation

Strains Conditions Concentration

(µmol per g dry wt of biomass)

NAD+ NADH

S-adhE xylose fermentation 9.83 ± 0.69 3.69 ± 0.19

S-nc 8.85 ± 0.23 5.24 ± 0.04

S-adhE glucose fermentation 8.88 ± 0.49 3.04 ± 0.26

S-nc 8.85 ± 0.24 3.24 ± 0.29

Strains Product yields (mole/mole substrate)

Acetate

consumption (mole per mole

xylsoe)

NAD+

generation

by acetate (mole per

mole xylsoe)

NAD+

generation

by glycerol (mole per

mole xylsoe)

Total NAD+

generation by

acetate+glycerol (mole per mole

xylsoe)

YetOH a Ygly Yxyl

S-adhE 1.145 0.158 0.073 0.078 0.156 0.158 0.314

S-nc 1.019 0.214 0.158 0.004 n. a. 0.214 0.214

Supplementary Table S4. Primers for PCR amplification of selected genes

Target genes Primer sequences

E. coli adhE Forward: GCCggatccAAAAATGGCTGTTACTAATGTCGCTG

Reverse: GCCctcgagTTAAGCGGATTTTTTCGCTTTTTTCTC

E. coli mphF Forward:

GCCggatccAAAAATGAGTAAGCGTAAAGTCGCCATATCGG

Reverse: GCCgtcgacTCATGCCGCTTCTCCTGCCTTG

E. coli eutE Forward:

GCCggatccAAAAATGAATCAACAGGATATTGAACAGG

Reverse: GCCctcgagTTAAACAATGCGAAACGCATCGAC

C.

beijerinckii

ALDH gene

Forward:

GCCggatccAAAAATGAGAGTTACGAATCCAGAAGAAT

Reverse: GCCctcgagTTATTTATTGCTGCCATTATATGAT

KanMX6 Forward: GCCgagctcAAATCCTTACGCATCTGTGCGGTAT

Reverse: AATGCCgagctcTTACGGGGCTGGCTTACTATGC

Note: nucleotide sequences in lowercase letters represent the restriction enzyme

recognition sites

Supplementary Note 1. Elementary flux mode analysis of the acetate reduction pathway

in S. cerevisiae

Recently, elementary flux mode (EFM) analysis has emerged as a powerful tool for

metabolic pathway analysis. An EFM is defined as a unique, minimal set of enzymes

(reactions) to allow steady state operation of a metabolic network with irreversible

reactions proceeding in the appropriate directions 57

. EFM analysis can decompose a

complex network of highly interconnected reactions into uniquely organized pathways

for characterization of cellular phenotypes and implementation of metabolic engineering

strategies. Based on the evaluation with acetoin, we explored the potential of using acetic

acid, an inhibitor in lignocellulosic hydrolysates, to improve ethanol yield from xylose

fermentation by serving as an external redox sink.

The results of the EFM analysis show that the acetate reduction pathway is operational in

yeast during xylose fermentation with a total of 677 calculated EFMs. Among these,

there were 0 anaerobic acetate-only consuming modes, 521 xylose-only consuming

modes, and 156 xylose and acetate consuming modes. In order to evaluate the effect of

the acetate reduction pathway, we calculated the theoretical ethanol yield and glycerol

yield for each of the xylose-only consuming modes and the xylose and acetate consuming

modes. Supplementary Fig. S3 shows box-and-whisker plots demonstrating the

beneficial potential of the acetate reduction pathway for increasing the ethanol yield and

reducing the glycerol accumulation during xylose fermentation. The median ethanol

yield when the acetate reduction pathway is active (0.479 g per g xylose) is significantly

higher than the median ethanol yield for the xylose only consuming modes (0.292 g per g

xylose). Similarly, the maximum achievable ethanol yield increases from 0.511 g per g

to 0.563 g per g when the acetate reduction pathway is operating. Also, the median

glycerol yield when the acetate reduction pathway is active (0 g per g xylose) is

significantly lower than the median yield for the xylose only consuming modes (0.344 g

per g xylose). The EFM analysis provided a theoretical basis for implementation of the

acetate reduction pathway in xylose-fermenting S. cerevisiae.

Supplementary Methods

The correlation between NAD+ regeneration and byproduct formation.

The Y parameter, NAD+ generated by external redox sink, was calculated from the

measured 2R,3R-butanediol production. The X parameter, decrease of byproduct

accumulation in each of the incubations, was calculated by subtracting the

xylitol+glycerol amount in the incubations of interest from that in the control incubation

without acetoin. Values of the two parameters for all the incubations at 24hr, 36hr, 48hr

and 60 hr were calculated and plotted. Linear regression generated a relationship Y=

1.37*X with R2=0.983. The xylitol and glycerol yields obtained from anaerobic xylose

fermentation by the strain SR8 were 0.116 g g-1

substrate and 0.128 g g-1

substrate,

respectively.

Elementary flux mode analysis.

A yeast core metabolic network was constructed for S. cerevisiae growing on xylose with

an acetate reduction pathway. The network was composed of 59 reactions which

included primary carbohydrate metabolism (glycolysis, TCA cycle, PP pathway,

gluconeogenesis) and energy metabolism (fermentation, respiration). Most importantly,

the reactions for acetate reduction were included in the model to examine the feasibility

of this pathway in engineered S. cerevisiae. A total of 47 internal metabolites

(maintained at steady state) and six external metabolites (sources and sinks) were defined

for the EFM calculation. The external metabolites included xylose, acetate, CO2,

glycerol, ethanol, and biomass. It should be noted that oxygen was not included as an

external metabolite meaning that we only considered the functionality of the acetate

reduction pathway under anaerobic conditions. The 677 total EFMs in the model were

calculated using METATOOL 5.1 with Matlab (2010, The Mathworks, Natick, MA,

USA).

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