Discovery of novel enzymes for the valorization of algal biomass:

from genomes of marine bacteria to blue biotechnology

Gurvan Michel

Marine Glycobiology group, UMR 8227

Me

-3OSO

OOSO

3-

O

O

OH

MeO

OSO3-

Polysaccharides

= ~50% algal biomass

Macroalgae: crucial role in the

coastal primary production

Support the coastal food webs:

from marine herbivorous animals

to human activities

(sea vegetables, hydrocolloids)

Me

-3OSO

OOSO

3-

O

O

OH

MeO

OSO3-

Polysaccharides

= ~50% algal biomass

Macroalgae: crucial role in the

coastal primary production

Marine heterotrophic bacteria:

key players in the recycling of algal biomass

Me

-3OSO

OOSO

3-

O

O

OH

MeO

OSO3-

Polysaccharides

= ~50% algal biomass

Macroalgae: crucial role in the

coastal primary production

Laminarin

b(1-3) b(1-3)

O

O

O H

O

O H

H O

O

O O H

O H

H O

O

O O H

O H

H O

O

O

O H

O H

O

Me

-3OSO

OOSO

3-

O

O

OH

MeO

OSO3-

a-1,3

a-1,4

agarose

k-carrageenan

i-carrageenan

l-carrageenan

b(1-4) a(1-3) O

OO

O

OH

O

OH OH

OH

O

OOO

OSO3-

O

OH

OH

OH

O

OOO

OSO3-

O

OH

OH

OSO3-

O

OSO3-

O

OH

O

OSO3-

OH

OHO

OSO3-

Alginates

L-Guluronic acids D-Mannuronic acids

a-1,4 b-1,4

O

OO O

O

O

OHOH

OH

HOOC

HOOC

O

HOOC

OH

OHO

OH

HOOC

OH

OH

Algal polysaccharides: a underexploited renewable biomass

Fucans

Laminarin

b(1-3) b(1-3)

O

O

O H

O

O H

H O

O

O O H

O H

H O

O

O O H

O H

H O

O

O

O H

O H

O

Me

-3OSO

OOSO

3-

O

O

OH

MeO

OSO3-

a-1,3

a-1,4

Fucans

agarose

k-carrageenan

i-carrageenan

l-carrageenan

b(1-4) a(1-3) O

OO

O

OH

O

OH OH

OH

O

OOO

OSO3-

O

OH

OH

OH

O

OOO

OSO3-

O

OH

OH

OSO3-

O

OSO3-

O

OH

O

OSO3-

OH

OHO

OSO3-

Huge chemical diversity

Algal sulfated polysaccharides have no equivalent in land plants

Diversity of the enzymes involved in their biosynthesis and their biodegradation

Algal polysaccharides: a underexploited renewable biomass

Zobellia galactanivorans: a model marine bacterium for

algae-bacteria interactions

• Bacteroidetes isolated in Roscoff on a red alga

• Degrade most algal polysaccharides

Genome sequencing (4738 genes, 5.5 MB)

• Large system for substrate detection and import

119 TonB-dependent receptors (TBDR) and 65 one/two-component systems

• Confirmation of the huge potential for polysaccharide degradation:

141 Glycoside hydrolases (GH) and 71 sulfatases !

• Bacteroidetes isolated in Roscoff on a red alga

• Degrade most algal polysaccharides

Zobellia galactanivorans: a model marine bacterium for

algae-bacteria interactions

Genome sequencing (4738 genes, 5.5 MB)

• Large system for substrate detection and import

119 TonB-dependent receptors (TBDR) and 65 one/two-component systems

• Confirmation of the huge potential for polysaccharide degradation:

141 Glycoside hydrolases (GH) and 71 sulfatases !

• Numerous putative operons specific for polysaccharide utilization

• Bacteroidetes isolated in Roscoff on a red alga

• Degrade most algal polysaccharides

Zg2624 Zg2615 Zg2613 Zg2614 Zg2616 Zg2617 Zg2618 Zg2619 Zg2620 Zg2621 Zg2622 Zg2623

Sugar kinase

transcriptional

factor UNK

SusD-like

TBDR

Sugar

dehydrogenase

KgdF-like

PL17 PL7

Sugar

permease

Sugar

dehydrogenase Sugar kinase

Zg2612

NRAMP

transporter

Zobellia galactanivorans: a model marine bacterium for

algae-bacteria interactions

Zg3628 AgaF

Zg3640 AgaG

Zg1017 AgaH

Zg3376 AgaI

Zg2600 AgaE

Zg4267 AgaC

Zg4203 AgaA

Zg3573 AgaB

Zg4243 AgaD

Zg236 CgkA

Zg2431 LamA

Zg367 LamB

Zg1010 LamC

Zg1014 LamD

Zg1021

Z3347

LicA Bl

74

100

96

63

59

95

67

48

46

33

74

37

38

41

0.5

Phylogenetic tree of the GH16 family from Zobellia

Allouch et al, (2003) JBC & ( 2004) Structure

Jam et al (2005) Biochem J

AgaB

AgaA

Barbeyron et al (1998)

Mol Biol Evol

Zg3628 AgaF

Zg3640 AgaG

Zg1017 AgaH

Zg3376 AgaI

Zg2600 AgaE

Zg4267 AgaC

Zg4203 AgaA

Zg3573 AgaB

Zg4243 AgaD

Zg236 CgkA

Zg2431 LamA

Zg367 LamB

Zg1010 LamC

Zg1014 LamD

Zg1021

Z3347

LicA Bl

74

100

96

63

59

95

67

48

46

33

74

37

38

41

0.5

GH16 X70 UNK

GH16

GH16 CBM6

GH16 CBM16

b-agarases

k-carrageenases

Phylogenetic tree of the GH16 family from Zobellia

Zg3628 AgaF

Zg3640 AgaG

Zg1017 AgaH

Zg3376 AgaI

Zg2600 AgaE

Zg4267 AgaC

Zg4203 AgaA

Zg3573 AgaB

Zg4243 AgaD

Zg236 CgkA

Zg2431 LamA

Zg367 LamB

Zg1010 LamC

Zg1014 LamD

Zg1021

Z3347

LicA Bl

74

100

96

63

59

95

67

48

46

33

74

37

38

41

0.5

Phylogenetic tree of the GH16 family from Zobellia

AgaD

Hehemann et al (2013) JBC

GH16 CBM6

Zg3628 AgaF

Zg3640 AgaG

Zg1017 AgaH

Zg3376 AgaI

Zg2600 AgaE

Zg4267 AgaC

Zg4203 AgaA

Zg3573 AgaB

Zg4243 AgaD

Zg236 CgkA

Zg2431 LamA

Zg367 LamB

Zg1010 LamC

Zg1014 LamD

Zg1021

Z3347

LicA Bl

74

100

96

63

59

95

67

48

46

33

74

37

38

41

0.5

GH16 PKD

GH16

GH16 UNK CBM42

GH16 CBM6

Laminarinases

Phylogenetic tree of the GH16 family from Zobellia

Zg3628 AgaF

Zg3640 AgaG

Zg1017 AgaH

Zg3376 AgaI

Zg2600 AgaE

Zg4267 AgaC

Zg4203 AgaA

Zg3573 AgaB

Zg4243 AgaD

Zg236 CgkA

Zg2431 LamA

Zg367 LamB

Zg1010 LamC

Zg1014 LamD

Zg1021

Z3347

LicA Bl

74

100

96

63

59

95

67

48

46

33

74

37

38

41

0.5

Phylogenetic tree of the GH16 family from Zobellia

GH16 PKD

GH16 CBM6

LamA_GH16

LamC_GH16

LamC_CBM6

Labourel et al (2014) JBC

Labourel et al (2015)

Acta Cryst D

Jam et al (2016)

FEBS Journal

Zg3628 AgaF

Zg3640 AgaG

Zg1017 AgaH

Zg3376 AgaI

Zg2600 AgaE

Zg4267 AgaC

Zg4203 AgaA

Zg3573 AgaB

Zg4243 AgaD

Zg236 CgkA

Zg2431 LamA

Zg367 LamB

Zg1010 LamC

Zg1014 LamD

Zg1021

Z3347

LicA Bl

74

100

96

63

59

95

67

48

46

33

74

37

38

41

0.5

GH16

GH16 UNK

GH16 UNK

GH16

GH16

GH16

A new sub-family

of GH16 ?

~25% identity with

AgaA and CgkA

Phylogenetic tree of the GH16 family from Zobellia

Zg3628 AgaF

Zg3640 AgaG

Zg1017 AgaH

Zg3376 AgaI

Zg2600 AgaE

Zg4267 AgaC

Zg4203 AgaA

Zg3573 AgaB

Zg4243 AgaD

Zg236 CgkA

Zg2431 LamA

Zg367 LamB

Zg1010 LamC

Zg1014 LamD

Zg1021

Z3347

LicA Bl

74

100

96

63

59

95

67

48

46

33

74

37

38

41

0.5

GH16

GH16 UNK

GH16 UNK

GH16

GH16

GH16

Phylogenetic tree of the GH16 family from Zobellia

Overexpression of

Zg1017 & Zg2600

Zg1017

Zg2600

Activity screening on cell wall extracts

from seaweeds

• Inactive on agarose and carrageenans

Zg1017

Zg2600

Activity screening on cell wall extracts

from seaweeds

L6S = L-galactose-6-sulfate

LA = 3,6-anhydro-L-galactose

Zg2600 (PorA) et Zg1017 (PorB) are the first beta-porphyranases

• Main end product: Porphyran disaccharide (L6S-G)

• Hydrolysis of beta-1,4 glycosidic linkage

a-1,3 b-1,4 • Inactive on agarose and carrageenans

PorB PorA

Crystal structure of the b-porphyranases PorA and PorB

Molecular surface (Basic residues are colored in dark blue)

W131

L6S L6S

G

G

-4

-3

-2 -1

E144

E139S

W56

H53

R133

R59

Catalytic residues

Structure of PorA in complex with porphyran

tetrasaccharide

R59

H53

R133

W67

R70

I141

R252

L6S MES

PorA PorB

L6S -2

LA -2

G -1 AgaB

No pocket in b-agarases:

Steric clash with L6S at subsite -2

agarases cannot degrade

porphyran

Conserved basic residues at subsite -2 are critical for

porphyran recognition

Hehemann et al (2010) Nature

Discovery of a new GH family in Zobellia

• 5 paralogous proteins distantly related to GH43 (~15% sequence identity)

• Always localized in gene clusters with CAZymes and sulfatases

• Zg3597 and Zg4663: soluble expressed in the MARINE-EXPRESS project

(Groisillier et al, 2010, Molecular Cell Factories)

• Purification and crystallization of Zg3597 and Zg4663

• Reducing sugar activity screening on our collection of algal polysaccharides

and cell wall extracts

Hypothesis: new GH specific for algal sulfated polysaccharides?

Unsuccessful…

?

?

?

Surrounded by b-agarases, b-porphyranases and b-galactosidases

Zg4663: a a-1,3-galactosidase specific for agarocolloids?

Neoagarobiose (DP2)

a-1,3

Test of the activity of Zg4663 on oligo-agars

released by the b-agarase AgaB

Thin layer chromatography

Neoagarobiose (DP2)

a-1,3

1121 (DP7)

3,6-anhydro-L-galactose

815 (DP5)

509 (DP3)

m/z (M+Na+)

Test of the activity of Zg4663 on oligo-agars

released by the b-agarase AgaB

Lane 2 and 4: addition of Zg4663

Size of the products determined by mass

spectrometry (MALDI-TOF)

Thin layer chromatography

• Zg4663 is a 1,3-a 3,6-anhydro-L-galactosidase (last step of agar degradation)

Neoagarobiose (DP2)

a-1,3

• Adopts a five-bladed b-propeller fold and forms a dimer by domain swapping

• Displays a zinc-dependent catalytic machinery

1121 (DP7)

3,6-anhydro-L-galactose

815 (DP5)

509 (DP3)

m/z (M+Na+)

• Zg4663 is a 1,3-a 3,6-anhydro-L-galactosidase (last step of agar degradation)

Rebuffet et al (2011) Environmental Microbiology

Alginate lyases

CAZy family PL5 PL6 PL7 PL14 PL15 PL17 PL18

Z. galactanivorans enzymes

- AlyA4 AlyA6

AlyA1 AlyA2 AlyA5

AlyA7 Zg4327 AlyA3 -

Different specificities?

Different modes of action?

Different biological functions?

Zobellia genome contains

7 putative alginate lyases

L-Guluronic acids D-Mannuronic acids

O

OO O

O

O

OHOH

OH

HOOC

HOOC

O

HOOC

OH

OHO

OH

HOOC

OH

OH

alyA3 SDR SusC-like SusD-like alyA2

GntR-like

permease

SDR

kdgK1

kdgF

alyA4 alyA5 alyA6

Alginate-related gene clusters?

alyA1

Transport

Outside

Periplasm

Cytoplasm

Sus-like system

permease

Regulation

GntR-like transcription regulator

Degradation

Alginate

4-deoxy-5-keto uronic acid

2-keto-3-deoxy-gluconate

ketodeoxyphosphogluconate

Alginate lyases

Gluconate dehydrogenases

(SDR)

kdgK

pair 1 pair 2 pair 3 G A G A G A

G : glucose A : alginate

Primer pairs

alyA3 SDR SusC-like SusD-like alyA2

GntR-like

permease

SDR

kdgK1

kdgF

alyA4 alyA5 alyA6

Primer pair 1 Primer pair 2

Primer pair 3

Gene expression with alginate vs glucose

Identification of two alginolytic operons!

Thomas et al (2012) Env Microbio

Characterization of alginate-responsive genes

Medium-throughput cloning strategy

Recombinant, soluble proteins

Confirmation of the enzymatic activity for 6 new proteins from Z. galactanivorans • Alginate lyases: AlyA1, AlyA4, AlyA5 and AlyA7 • 2-dehydro-3-deoxygluconokinase: Zg2614 • 2-dehydro-3-deoxy-D-gluconate 6-dehydrogenase: Zg2622

Characterization of alginate-responsive genes

Medium-throughput cloning strategy

Recombinant, soluble proteins

AlyA1 and AlyA5 Two new lyases from the PL7 family

-1 +1 +2 +3

-1 +1 +2 +3

AlyA1: endo-lyase

AlyA5: exo-lyase

Thomas et al, (2013) JBC

Conclusions & perspectives

Conclusions

• Algae-associated bacteria are essential sources of novel marine enzymes

• A (semi)-rational strategy for the discovery of novel enzymes: combination of phylogenomics, comparative genomics and biochemical approaches

• Importance of libraries of natural polysaccharides / oligosaccharides for the screening of real functions

Conclusions & perspectives

Conclusions

• Algae-associated bacteria are essential sources of novel marine enzymes

• A (semi)-rational strategy for the discovery of novel enzymes: combination of phylogenomics, comparative genomics and biochemical approaches

• Importance of libraries of natural polysaccharides / oligosaccharides for the screening of real functions

Perspectives: start of two new projects in 2015

ALGOLIFE: processing macroalgae for high value-added human and animal

health/nutrition products

Thank you!

Marine Glycobiology Group

Aurore Labourel Jan-Hendrik Hehemann

François Thomas

Etienne Rebuffet