Reticulate evolution in the Saccharomyces genus and

repeating it for the bioethanol industry

David Peris, Kayla Sylvester, Maria Sardi, William Alexander,

Diego Libkind, Paula Gonçalves José Sampaio,

Lucas Parreiras, Trey Sato, Chris Hittinger

Diversity and Biotechnology

Characterization of the diversity in the Saccharomyces genus

S. mikatae

S. arboricolus

S. cerevisiae

Patagonia A

Patagonia B/Tibet* (Lager)

West China

0.8%

3.9%

Holartic*/South America A

South America B

Australasia

4.4%1%

S. eubayanus

S. uvarum

S. kudriavzevii3.5%

1.2% Europe*

East Asia A

East Asia B

West/Eurasia A

America B

America C

Far East

>1%

2.5-3.5%1.8%

S. paradoxus

China VChina IV

China IIIChina II

China I

Wine/European*China VI-VIII*

SakeNorth AmericanMalaysian

West African

<1%

0.4%

Sichuan

Diversity and Biotechnology

Characterization of the diversity in the Saccharomyces genus

Application of targeted traits to improve bioethanol production

S. mikatae

S. arboricolus

S. cerevisiae

Patagonia A

Patagonia B/Tibet* (Lager)

West China

0.8%

3.9%

Holartic*/South America A

South America B

Australasia

4.4%1%

S. eubayanus

S. uvarum

S. kudriavzevii3.5%

1.2% Europe*

East Asia A

East Asia B

West/Eurasia A

America B

America C

Far East

>1%

2.5-3.5%1.8%

S. paradoxus

China VChina IV

China IIIChina II

China I

Wine/European*China VI-VIII*

SakeNorth AmericanMalaysian

West African

<1%

0.4%

Sichuan

Bioethanol pipeline

Cropping Systems Pretreated Biomass Hydrolysate

DECONSTRUCTION PLANTS CONVERSION

Bioethanol

Hydrolysate Challenges – C5 sugars Proteins,

Oils, Ash (0-2%)

Hemicellulose

(19-34%)

Lignin

(21-32%)

Cellulose

(33-51%) Glucose

Xylose Arabinose

Sugars (C6/C5)

Mannose Galactose

Hydrolysate Challenges - Lignotoxins Proteins,

Oils, Ash (0-2%)

Hemicellulose

(19-34%)

Lignin

(21-32%)

Cellulose

(33-51%) Glucose

Xylose

HMF

Ferulic

acid

p-coumaric

acid

Feruloyl amide

Sodium

acetate

Acetamide

Sugars (C6/C5) Lignotoxins

Mannose Galactose

Arabinose

Bioethanol industry workhorse

S. cerevisiae chassis strains ~0.4% nucleotide

diversity

Trey

Sato

Audrey

Gasch

AFEX Corn Stover

Hydrolysate

(ACSH)

S. cerevisiae chassis strains ~0.4% nucleotide

diversity

Trey

Sato

Audrey

Gasch

Chassis Strain

Engineering

Evolution

Y128 Y101

AFEX Corn Stover

Hydrolysate

(ACSH)

Chassis strains still require improvements

~0.4% nucleotide

diversity

Trey

Sato

Audrey

Gasch

Chassis Strain

Engineering

Evolution

Y128

Xylose

Lignotoxins

Y101

AFEX Corn Stover

Hydrolysate

(ACSH)

S. cerevisiae

S. mikatae

S. paradoxus

S. kudriavzevii

S. arboricolus S. uvarum S. eubayanus

0.03

COX3 (mtDNA gene)

Saccharomyces is highly diverse

S. mikatae

S. paradoxus

S. kudriavzevii

S. arboricolus S. uvarum S. eubayanus

0.03

Saccharomyces is highly diverse

COX3 (mtDNA gene)

S. cerevisiae

Harnessing Saccharomyces diversity

S. mikatae

S. paradoxus

S. kudriavzevii

S. arboricolus S. uvarum S. eubayanus

0.03

COX3 (mtDNA gene)

S. cerevisiae

Hydrolysate tolerance varies in Saccharomyces

-2.0

-1.8

-1.6

-1.4

-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6lo

g2 (μstrain

/μref Y

101)

32 28 85 47 135 130 GLBRCY101

GLBRCY128 N=

Aerobic

28ºC

Public Goods for innovation S. paradoxus

S. mikatae

ALD

Xylose transporter

ADH

Lignotoxin tolerance

Chassis Strain

Super Fuel Strain

We need to sequence the genomes.

Public Goods for innovation S. paradoxus

S. mikatae

ALD

Xylose transporter

ADH

Lignotoxin tolerance

Chassis Strain

Super Fuel Strain

We need to sequence the genomes.

Nascent techniques for the detection of

differential traits.

Public Goods for innovation S. paradoxus

S. mikatae

ALD

Xylose transporter

ADH

Lignotoxin tolerance

Chassis Strain

Super Fuel Strain

We need to sequence the genomes.

Nascent techniques for the detection of

differential traits.

Cloning problems.

Public Goods for innovation S. paradoxus

S. mikatae

ALD

Xylose transporter

ADH

Lignotoxin tolerance

Chassis Strain

Super Fuel Strain

Gonzalez et al. 2008

Dunn et al. 2008

Peris et al. 2012a,b,c

Libkind et al. 2011

Almeida et al 2014

Reticulate evolution: hybridization

S. pastorianus

S. paradoxus

S. mikatae

S. arboricolus

S. kudriavzevii

S. uvarum

S. cerevisiae

S. eubayanus

S. bayanus

Hybridization by mass mating (nxn)

S. cerevisiae

Y101

X

S. mikatae

S. cerevisiae

Y101

X

S. kudriavzevii

S. cerevisiae

Y101

X

S. uvarum

S. cerevisiae

Y128

X

S. mikatae

S. cerevisiae

Y128

X

S. kudriavzevii

S. cerevisiae

Y128

X

S. uvarum

yHDPG1 yHDPG2 yHDPG5 yHDPG6 yHDPG7 yHDPG8

Hybrids maintain the tolerance to lignotoxins

-1.0

-0.9

-0.8

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

S. cerevisiae

X

S. mikatae

S. cerevisiae

X

S. kudriavzevii

S. cerevisiae

X

S. uvarum

yHDPG1 yHDPG5 yHDPG7

Y101

log

2 (μstrain

/μref Y

101)

Aerobic

28ºC

S. mikatae

S. kudriavzevii

S. uvarum

Y101

-1.0

-0.9

-0.8

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

Hybrids maintain the tolerance to lignotoxins

S. mikatae

S. kudriavzevii

S. uvarum

Y128

S. cerevisiae

X

S. mikatae

S. cerevisiae

X

S. kudriavzevii

S. cerevisiae

X

S. uvarum

yHDPG2 yHDPG6 yHDPG8

Y128

Aerobic

28ºC lo

g2 (μstrain

/μref Y

101)

Evolved hybrids improve xylose consumption

R1 R9 …

yHDPG5

S. cerevisiae

Y101

X

S. kudriavzevii

IFO1802

50G

Aerobic

30ºC

Evolved hybrids improve xylose consumption

R1 R3 R5

Evolution round

%

Consumption g/L

(EtOH)

R7 R9

(50G)

R1 R9 …

0

5

10

15

20

25

30

35

40

0

10

20

30

40

50

60

70

80

90

100

Glc

Xyl

EtOH

50G

yHDPG5

Aerobic

30ºC

S. cerevisiae

Y101

X

S. kudriavzevii

IFO1802

0

5

10

15

20

25

30

35

40

0

10

20

30

40

50

60

70

80

90

100

Glc

Xyl

EtOH

Conclusions

We observed substantial variation in the Saccharomyces

genus.

0

5

10

15

20

0 48 96 144 192 240 288 336 384

Conclusions

We observed substantial variation in the Saccharomyces

genus.

Diversity can provide genes to improve many industrial

processes.

0

5

10

15

20

0 48 96 144 192 240 288 336 384

Conclusions

We observed substantial variation in the Saccharomyces

genus.

Diversity can provide genes to improve many industrial

processes.

S. mikatae is a promising species that might contain

lignotoxin tolerance genes.

Lignotoxin tolerance

0

5

10

15

20

0 48 96 144 192 240 288 336 384

Conclusions

We observed substantial variation in the Saccharomyces

genus.

Diversity can provide genes to improve many industrial

processes.

S. mikatae is a promising species that might contain

lignotoxin tolerance genes.

Hybridization is a promising shortcut for improving chassis

strains.

Lignotoxin tolerance

0

5

10

15

20

0 48 96 144 192 240 288 336 384

Acknowledgements

Chris Hittinger

Kayla Sylvester

William Alexander

Emily Baker

Meihua Kuang

Hittinger Lab Members

Wild YEAST program

Trey Sato

Li Hinchman

Lucas Parreiras

Jeff Piotrowski

Diego Libkind

Jose Paulo Sampaio

Paula Gonçalves

Christian Landry

Jean-Baptiste Leducq

Justin Fay

Katie Hyma

Fengyan Bai

Qi Ming Wang

Yaoping Zhang

Alex Reau

Haibo Li

David Benton

Yury Bukhman

Oleg Moskvin

HPLC Service

Mick McGee

Audrey Gasch

Maria Sardi

UW & GLBRC Collaboration

Thank you

Poster: 349A