FAC U LTY O F SC I EN C E
UNIVERSITY OF COPENHAGEN
Novel Algae Degrading Enzymes from Metagenomes
and Sea Urchin Microbiota
Mikkel
*
Schultz-Johansen ,
Mikkel Andreas Glaring, Jan Kjølhede Vester and Peter Stougaard
University of Copenhagen, Department of Plant and Environmental Sciences, Section for Genetics and Microbiology. *: [email protected].
Introduction
Seaweeds and other algae possess industrial application potentials in biofuels and contain bioactive compounds that can be used in
nutraceuticals and pharmaceuticals. Novel enzymes capable of hydrolysing specific algal cell wall polysaccharides such as agar, alginate and
carrageenan, may be used to optimize the extraction of fermentable sugars and valuable biomolecules from algae. In the search for novel
algae degrading enzymes we have analysed metagenomes obtained from bacteria growing in enrichment cultures with seaweed and found
several putative genes coding for hydrolytic enzymes, which act on algal cell wall polysaccharides. Algae-eating animals, such as sea urchins,
may possess a microflora optimized for hydrolysis of algal cell wall material. Investigation of bacteria isolated from the digestive system of sea
urchins resulted in the identification of enzyme producing bacterial isolates capable of degrading agar, alginate and carrageenan.
Methods
B) Isolation and screening of bacteria from sea urchin microbiota
A) Metagenomics approach
The culture media contained an extract of
brown algae or the pure algal cell wall
polysaccharides: alginate, fucoidan or
furcelleran as the sole carbon source.
Four bacterial enrichment
cultures were established
on brown algae.
Cultivable bacteria were
isolated from sea urchin
gut and intestines.
Total DNA was extracted
and metagenomic data was
obtained and analysed.
Bacterial isolates were screened
for production of algal cell wall
degrading enzymes.
Sea urchins were collected
and the digestive tract was
removed.
Results
A) Metagenomics approach
Table 1: Amount of putative algae degrading enzyme genes in metagenomes based on HMM search against algae polysaccharidase containing CAZy protein families (Cut off value: ≤10E-5).
Symbols indicate that CAZy family contains proteins with the following activities: ■ Agarase ▲ Alginate lyase ▬ Fucoidanase ♦ Carrageenase ● Porphyranase ♠ Laminarinase
GH16 ■ ♦ ● ♠
Metagenome
CAZy Family 
GH50 ■ GH82 ♦
GH86 ■ ●
GH96 ■
GH107 ▬
GH118 ■
PL5 ▲ PL6 ▲ PL7 ▲ PL14 ▲ PL15 ▲ PL17 ▲ PL18 ▲
Brown algae extract
10
7
1
2
0
4
0
5
14
23
2
2
13
1
Alginate
4
7
0
0
0
1
0
1
14
14
0
1
7
0
Fucoidan
3
17
0
0
0
0
0
6
1
12
0
1
2
0
Furcelleran
17
9
4
1
0
0
0
4
7
5
0
2
3
0
• Analysis of metagenomes revealed several genes encoding putative algae
degrading enzymes, many of which were novel (protein identity <80%). One of the
predicted proteins (Car4), which was related to the GH16 Family of glycoside
hydrolases, shared 59 % identity with an annotated kappa-carrageenase from
Catenovulum agarivorans. By cloning the gene encoding Car4 into E. coli we have
been able to produce a novel recombinant enzyme with activity towards kappacarrageenan. This enzyme is currently being characterised.
Figure 1: The closest relatives of Car4 based on a BLASTp similarity search.
Outgroup: kappa-carrageenase from P. carrageenovora
B) Isolation and screening of bacteria from sea urchin microbiota
Table 2: Phylogenetic affiliation of algae degrading isolates from sea urchins based on 16S rRNA analysis.
Isolate
Closest relative % identity Base pairs sequenced Agarase Carrageenase Alginate lyase
SpA1
Colwellia asteriadis
97
997
+
-
+
SpF5
Cellulophaga baltica
99
1012
-
+
+
SpF6
Formosa algae
99
1093
-
-
+
SpF8
Algibacter lectus
98
1108
-
+
+
• Bacteria were isolated from the digestive tract of sea urchins. The isolates
were capable of degrading one or more algal polysaccharides including
agar, alginate and carrageenan.
Figure 2: Alginate lyase activity of bacterial isolates SpF5 and SpF6
grown on alginate containing plate medium. Alginate degradation
was visualised by staining with cetyl pyridinium chloride
Outlook
Future work
Genes encoding novel algae degrading enzymes were obtained by metagenomic sequencing of enrichment cultures. Isolation of bacteria from sea urchins, that degraded
agar, alginate and carrageenan, suggest that sea urchins could be a source of algae degrading enzymes. Sequencing of 16S rRNA amplicons and metagenomic studies
of total DNA from sea urchin microbiota, might provide more insight into the bacterial population inhabiting sea urchin digestive tracts and their role in algae degradation.
Novel enzymes identified from metagenomes and sea urchin microbiota will be cloned and expressed in a suitable host organism using traditional PCR dependent
methods as well as high throughput screening of metagenomic expression libraries. Characterisation of resulting recombinant enzymes will be performed.