The document summarizes research on using experimental evolution and hybridization of Saccharomyces yeasts to develop strains for improved cellulosic biofuel production. Key points include: - The diversity of Saccharomyces yeasts was mapped and certain lineages were selected for hybridization based on traits like tolerance. - Hybrids were generated between S. cerevisiae (the chassis strain) and other species and then experimentally evolved over 50 generations. - The evolved hybrid strains showed improved utilization of xylose compared to ancestor strains, producing similar ethanol levels without accumulating byproducts. - Genome analysis found the evolved hybrids were mostly triploid and had chromosomal rearrangements replacing parent genes. - Further work aims to

1. Mining Saccharomyces diversity and experimental
evolution for cellulosic biofuel production
David Peris, Postdoctoral Research Associate
16th July 2016

2. Surfing the diversity of Saccharomyces genus
Phylogenomic tree
23/30 Lineages represented
Whole Genome sequences
Lineages not available
S. cerevisiae
S. paradoxus
S. mikatae
S. arboricola
S. kudriavzevii
S. eubayanus
S. uvarum
North America B
North AmericaC
Far East
North America A
Europe-A
Asia- A
Asia- B
Asia- A
Europe
Near East
Asia- B
Oceania
Asia- A
SouthAmerica A
Holarctic NA
Holarctic EU
South America B
Australasia
Patagonia B
Holarctic
Patagonia A
Nakaseomycescastellii (outgroup)
0.02
West China
Sichuan

3. Surfing the diversity of Saccharomyces genus
Langdon Q. P2018/C
Population Structure Seub
Peris et al. 2014 Mol Ecology
Peris and Langdon et al. 2016 PLOS Genetics
S. cerevisiae
S. paradoxus
S. mikatae
S. arboricola
S. kudriavzevii
S. eubayanus
S. uvarum
North America B
North AmericaC
Far East
North America A
Europe-A
Asia- A
Asia- B
Asia- A
Europe
Near East
Asia- B
Oceania
Asia- A
SouthAmerica A
Holarctic NA
Holarctic EU
South America B
Australasia
Patagonia B
Holarctic
Patagonia A
Nakaseomycescastellii (outgroup)
0.02
West China
Sichuan
Baker E. Y3125/B
Lager domestication of Seub
Baker et al. 2015 MBE

4. Surfing the diversity of Saccharomyces genus
S. cerevisiae
S. paradoxus
S. mikatae
S. arboricola
S. kudriavzevii
S. eubayanus
S. uvarum
North America B
North AmericaC
Far East
North America A
Europe-A
Asia- A
Asia- B
Asia- A
Europe
Near East
Asia- B
Oceania
Asia- A
SouthAmerica A
Holarctic NA
Holarctic EU
South America B
Australasia
Patagonia B
Holarctic
Patagonia A
Nakaseomycescastellii (outgroup)
0.02
West China
Sichuan

5. AFEX-Corn Stover (ACSH) Challenges
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

6. Testing hybridization method
Gonzalez et al. 2008
Dunn et al. 2008
Peris et al. 2012a,b,c
Libkind et al. 2011
Almeida et al 2014
S. pastorianus
S. paradoxus
S. mikatae
S. arboricolus
S. kudriavzevii
S. uvarum
S. cerevisiae
S. eubayanus
S. bayanus

7. Picking our chassis strain
Y732n
CHASSIS
The most tolerant of S. cerevisiae
Engineered with xylose utilization genes
Wohlbach et al. 2009 PNAS
Sato et al. 2013 AEM

8. Generation of hybrids between chassis and reference species
Y101 n n
S.mikatae S.kudriavzevii
X
R1 R9…
50 Generation
30ºC
14days
Y732n
Growth rate better than both parents
CHASSIS
tolerant
xylose utilizattion

9. Evolved hybrids utilize more xylose than ancestors
Y101 n n
S.mikatae S.kudriavzevii
X
D-Xylose
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
Acetate
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
Xylitol
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
Ethanol
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
T4
(14 days)
g/L
CHASSIS
ANCESTOR
EVOLVED
ANCESTOR
EVOLVED
D-xylose

10. Evolved hybrids do not accumulate xylitol or acetate
Y101 n n
S.mikatae S.kudriavzevii
X
D-Xylose
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
Acetate
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
Xylitol
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
Ethanol
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
g/L
D-xylosexylitolAcetate
CHASSIS
ANCESTOR
EVOLVED
ANCESTOR
EVOLVED
CHASSIS
ANCESTOR
EVOLVED
ANCESTOR
EVOLVED
CHASSIS
ANCESTOR
EVOLVED
ANCESTOR
EVOLVED
T4
(14 days)

11. Ethanol levels of evolved hybrids similar to Scer
Y101 n n
S.mikatae S.kudriavzevii
X
D-Xylose
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
Acetate
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
Xylitol
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
Ethanol
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
g/L
CHASSIS
ANCESTOR
EVOLVED
ANCESTOR
EVOLVED
D-xylosexylitolAcetateEthanol
CHASSIS
ANCESTOR
EVOLVED
ANCESTOR
EVOLVED
CHASSIS
ANCESTOR
EVOLVED
ANCESTOR
EVOLVED
CHASSIS
ANCESTOR
EVOLVED
ANCESTOR
EVOLVED
T4
(14 days)

12. Improvement by gross chromosomal rearrangements
Y101 n n
S.mikatae S.kudriavzevii
X
D-Xylose
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
Acetate
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
Xylitol
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
Ethanol
Sc_GLBRCY73
Anc-ScxSm_yHDPN1
Anc-ScxSk_yHDPN5
Evol-ScxSm_yHDPN399
Evol-ScxSk_yHDPN379
g/L
3 2 2
1
2 2 2 2 2
1
2 2 2 2 2 2
0
2 2 2
1 1 1 1
2 2 2
1 1 1
2
1
I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI
0
1
2
3
4
5
0
1
2
3
4
5
RC/MRC
I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI
3
0
4
copies
3
copies Translocations
Replacement of
one parent
genes
CHASSIS
ANCESTOR
EVOLVED
ANCESTOR
EVOLVED
D-xylosexylitolAcetateEthanol
CHASSIS
ANCESTOR
EVOLVED
ANCESTOR
EVOLVED
CHASSIS
ANCESTOR
EVOLVED
ANCESTOR
EVOLVED
CHASSIS
ANCESTOR
EVOLVED
ANCESTOR
EVOLVED
T4
(14 days)
Mostly triploid (3n)

13. Using the full genomic diversity of species with HyPr
S.mikatae
S.paradoxus S.eubayanus
S.kudriavzevii
S.arboricola
2n 2nX
S.cerevisiae
GLBRCY73
Alexander et al 2016 FGB
Hybrid Production
(HyPr)
S.uvarum

14. Selecting the best strains from Saccharomyces spp.
S.mikatae
S.paradoxus
S.uvarum
S.eubayanus
S.kudriavzevii
S.arboricola
2n 2nX
S.cerevisiae
GLBRCY73
Moriarty R. Y3131/B
Mining Saccharomyces diversity
Alexander et al 2016 FGB
Hybrid Production
(HyPr)
Screening of 511 Saccharomyces strains
Lignotoxin
tolerance
Xylose
utilization
Good levels
of
ethanol

15. Using the full genomic diversity of species with HyPr
S.mikatae
S.paradoxus S.eubayanus
S.kudriavzevii
S.arboricola
2n 2nX
S.cerevisiae
GLBRCY73
Alexander et al 2016 FGB
Hybrid Production
(HyPr)
S.uvarum
Ethanol
Isobutanol
Production of interesting compounds
Other applications

16. Thank you
Chris Hittinger
Ryan V. Moriarty: Y3131/B
Kayla Sylvester
William Alexander
Emily Baker: Y3125/B
Meihua Kuang
Quinn Langdon: P2018/C
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
Guillaume Charron
Justin Fay
Katie Hyma
Fengyan Bai
Qi Ming Wang
Yaoping Zhang
Alex Reau
Haibo Li
David Benton
Yury Bukhman
HPLC Service
Mick McGee
Audrey Gasch
Maria Sardi
UW & GLBRC Collaboration