Artificial hybrids & biofuels

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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

description

In the last decade, the application of new molecular techniques has helped to identify Saccharomyces species and suggested many types of reticulated evolutionary events, such as hybridization, interspecies recombination, introgression, horizontal gene transfer, and admixture. Many strains isolated from fermentative environments, such as S. bayanus, which was isolated from beer or fermented beverages, and S. pastorianus, used in making lager beer, have been misidentified as distinct species when they have chromosome contributions from two or more natural Saccharomyces species. These hybridization events likely occurred in the stressful conditions found in industrial environments where hybrids were better suited. These hybrids do not have unique origins, suggesting that hybridization has a clear selective advantage. We have recently described two well-differentiated Patagonian populations of S. eubayanus, the wild contributor to S. pastorianus (S. cerevisiae x S. eubayanus) lager-brewing strains. The application of the state-of-the-art phylogenetic methods (Supernetworks and Bayesian concordance factor analysis) illuminated many reticulation events in S. eubayanus outside of Patagonia and uncovered the relationship between one population of S. eubayanus from Patagonia and its counterpart in S. bayanus and S. pastorianus. We also recently isolated S. eubayanus strains from Sheboygan, WI that originated by intraspecific hybridization (admixture) of the two Patagonian populations, and we used supernetworks to graphically represent the hybridization and recombination in S. bayanus and S. pastorianus from the brewing industry. With the knowledge gained by understanding the process of hybrid evolution that occurred in winemaking and brewing environments, we are developing new methods to generate artificial hybrids to repeat the hybridization process for the bioethanol industry.

Transcript of Artificial hybrids & biofuels

Page 1: Artificial hybrids & biofuels

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

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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

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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

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Bioethanol pipeline

Cropping Systems Pretreated Biomass Hydrolysate

DECONSTRUCTION PLANTS CONVERSION

Bioethanol

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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

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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

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Bioethanol industry workhorse

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S. cerevisiae chassis strains ~0.4% nucleotide

diversity

Trey

Sato

Audrey

Gasch

AFEX Corn Stover

Hydrolysate

(ACSH)

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S. cerevisiae chassis strains ~0.4% nucleotide

diversity

Trey

Sato

Audrey

Gasch

Chassis Strain

Engineering

Evolution

Y128 Y101

AFEX Corn Stover

Hydrolysate

(ACSH)

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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)

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S. cerevisiae

S. mikatae

S. paradoxus

S. kudriavzevii

S. arboricolus S. uvarum S. eubayanus

0.03

COX3 (mtDNA gene)

Saccharomyces is highly diverse

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S. mikatae

S. paradoxus

S. kudriavzevii

S. arboricolus S. uvarum S. eubayanus

0.03

Saccharomyces is highly diverse

COX3 (mtDNA gene)

S. cerevisiae

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Harnessing Saccharomyces diversity

S. mikatae

S. paradoxus

S. kudriavzevii

S. arboricolus S. uvarum S. eubayanus

0.03

COX3 (mtDNA gene)

S. cerevisiae

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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

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Public Goods for innovation S. paradoxus

S. mikatae

ALD

Xylose transporter

ADH

Lignotoxin tolerance

Chassis Strain

Super Fuel Strain

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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

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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

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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

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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

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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

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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

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-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)

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Evolved hybrids improve xylose consumption

R1 R9 …

yHDPG5

S. cerevisiae

Y101

X

S. kudriavzevii

IFO1802

50G

Aerobic

30ºC

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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

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Conclusions

We observed substantial variation in the Saccharomyces

genus.

0

5

10

15

20

0 48 96 144 192 240 288 336 384

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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

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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

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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

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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

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Thank you

Poster: 349A