Features
Metabolic pathways
The shaping and use of metabolic pathways in algae
Green factories
Steven G. Ball (University of Lille-1) and Gilles Peltier (CEA-Cadarache, France)
The complex endosymbiotic history of algal plastids has generated a high degree of diversity within
their metabolic pathways. The shaping and merging of pathways from various combinations of
hosts and endosymbionts is responsible for important biochemical innovations such as those
exemplified by the emergence of starch metabolism. Green algae, such as Chlamydomonas
reinhardtii, contain an oxygen-sensitive high‑specific‑activity hydrogenase that, in special
circumstances, can generate molecular hydrogen directly from photosynthesis, or indirectly through the storage of photosynthetic energy in starch. The challenge now facing biochemists studying these pathways is to make use of these organisms to produce
molecular hydrogen in a sustainable and efficient fashion.
Key words: biotechnology, Chlamydomonas
reinhardtii, endosymbiosis,
hydrogenase, storage
polysaccharide metabolism
Algae encompass a diverse array of
organisms that cannot be consid‑
ered as a single group. They are thus
considered polyphyletic (composed
of several groups of distinct origin) as
opposed to the monophyletic groups of animals, fungi
or terrestrial plants. In fact, they resist all attempts at
a simple definition. They were previously considered
as chlorophyll a‑containing organisms able to perform
oxygenic photosynthesis without terrestrial plant
features, such as roots, stems and leaves. However,
this definition is unsatisfactory, as a growing number
of authors consider organisms derived from classical
algae and which have lost the ability to photosynthe‑
size as ‘white’ algae. In addition, bona fide multicellular
red and brown algae often exhibit highly specialized
and differentiated structures that are just as complex
as those that characterize terrestrial plants.
Despite their polyphyletic nature, algae display a
common history which parallels that of the plastids
they all contain1. The story begins with internalization
by a heterotrophic eukaryotic ancestor of a cyanobac‑
terium. This primary endosymbiosis of a blue–green
alga, the only prokaryotic organism considered as
such, was followed by the emergence of three lineages
that collectively form the Archaeplastida. The event
occurred only once, possibly over a billion years
ago, and generated the Chloroplastida (green algae),
the Rhodophyceae (red algae) and the glaucophytes.
Glaucophyta define,single‑cell freshwater algae which
harbour plastids that still contain the peptidoglycan
wall of their cyanobacterial ancestors.
Had the endosymbiosis story stopped there,
eukaryotic algae would have been considered mono‑
phyletic. However, more complexity was to follow, as
2 June 2009 © 2009 The Biochemical Society
unicellular green and red algae themselves became
internalized by other heterotrophic eukaryotes to
generate the secondary plastids, a process known as
secondary endosymbiosis1. Secondary plastids are
typically composed of four membranes rather than
the two that characterize the primary plastids of
Archaeplastida. The two outer membranes of secondary
plastids correspond on the one hand to the phagocytic
vacuole of the heterotrophic eukaryotic host respon‑
sible for the internalization of the red or green alga,
and on the other hand to the plasma membrane of the
alga. The two inner membranes correspond to those of
the ancient rhodoplast or chloroplast in the case of a
secondary endosymbiosis, involving a red or a green
alga respectively. The huge diversity of organisms that
was generated is briefly outlined in Figure 1.
Metabolic pathways were deeply influenced by this
complex endosymbiotic history, which resulted in the
presence within algae of both a greater diversity of
biochemical pathways and often a greater complexity
within the architecture of the pathways themselves. The
diversity stems from the contribution of several distinct
genomes in the emergence of primary or secondary
endosymbiosis lines, while the complexity comes from
the merging of pathways common to the partners of
these various endosymbioses. These mergings occurred
during the complex evolutionary process that changed
a recently acquired endosymbiont into a true plastid.
We will very briefly illustrate these aspects by examin‑
ing as an example the pathway of starch metabolism
in microalgae and then turn our attention to the
exploitation of the diversity of pathways present in
Chloroplastida and in particular in the only model
organism intensively studied in algae: the unicellular
green alga C. reinhardtii
Metabolic pathways
The making of storage polysaccharide
pathways in algae
Glaucophytes
Primary
Endosymbiosis
Starch
Glycogen
Green plants
(Metaphytes)
Red Algae
Cryptophytes
Euglenophytes
neag
Red li
e
Red lineage
Green lineage
The algae can be divided into two lineages according to
storage polysaccharide metabolism: the starch and the
β-glucan (paramylon, laminarans) accumulators2. This
probably reflects the nature of the storage polysac‑
charide metabolic pathway present in the cytoplasm of
the heterotrophic host in a particular endosymbiotic
event. Eukaryotes in general can indeed be subdivided
into glycogen (α-glucan) or β-glucan accumulators. If
both partners of a particular endosymbiosis synthesize
the same types of polymers (for instance glycogen or
glycogen and starch) then the probability that these
pathways will merge is high. This is, seemingly, what
happened after primary endosymbiosis of the plastid.
All three Archaeplastid lines that resulted from prima‑
ry endosymbiosis proved to be starch accumulators.
Starch defines a mixture of amylose and amylopec‑
tin in a semi-crystalline solid state, whereas glycogen
defines water‑soluble particles consisting of a unique
polysaccharide fraction. Nevertheless, their metabolic
pathways are closely related since they both are made
of α-1,4‑linked glucans branched in α-1,6 position. An
interesting property of starch is that its distribution
seems restricted to the Archaeplastida, to some (but
not all) of their secondary endosymbiosis derivatives
(see Figure 1) and to a particular subgroup of unicel‑
lular nitrogen‑fixing cyanobacteria2,3.
The enzymes of the starch pathway in all Archae‑
plastida define a similar type of mixture of enzymes,
of either cyanobacterial or eukaryotic origin, which is
common to glaucophytes, and to red and green algae.
This suggests that both partners of endosymbiosis
synthesized related storage polysaccharides and that
the three lineages are indeed derived from a common
ancestor. Among the Archaeplastida, both red algae
and glaucophytes synthesize starch in the cytoplasm,
whereas all green algae (Chloroplastida) accumulate
these polymers within their plastids3,4. These and many
other reasons make a strong case for the loss of starch
metabolism by the endosymbiont at an early stage and
the presence of cytosolic starch within the common
ancestor of all Archaeplastida3.
Bioinformatic analysis of genomes from starchaccumulating cyanobacteria, red algae or their second‑
ary endosymbiosis apicomplexa parasite derivatives,
yields a pathway composed of at most 12 genes related
to those that are used for bacterial or eukaryotic gly‑
cogen metabolism. Interestingly, the Rhodophyceae
show an essentially complete set of eukaryotic glyco‑
gen metabolism, but only a few genes originating from
cyanobacterial pathways, precisely those suspected
to be responsible for the accumulation of starch in‑
N : Nucleus
Nm : Nucleomorph
Features
Dinoflagellates
Chlorarachniophytes
Secondary
Endosymbiosis
Apicomplexans
Heterokonts
Haptophytes
Figure 1. The complex endosymbiotic history of algae starts with a unique event: primary
endosymbiosis of a blue–green alga (displayed in blue) by a heterotrophic eukaryotic ancestor generating green algae (Chloroplastida), red algae (Rhodophyceae) and glaucophytes.
In turn, the Chloroplastida and Rhodophyceae were internalized by several distinct heterotrophic eukaryotes. The latter are not displayed, since the number of secondary events is, at
present, controversial. There is, however, an agreement on a minimum of three distinct events.
In two distinct events, a degenerate nucleus of the former green (Chlorarachniophytes) or red
(cryptophytes) still remains between the second and third membrane of the secondary
plastid. Apicomplexan parasites contain a non photosynthetic secondary plastids. Loss of
photosynthesis occured independently in several algal lineages. Non‑parasitic heterotrophic
algae are often referred to as the white algae.
stead of glycogen. This comes in striking contrast with the bioinformatic analysis
results yielded by the Chloroplastida, where a complex pathway of over 40 genes
is evidenced for starch synthesis and degradation within plastids4. However, this
complexity is largely due to duplications and subfunctionalizations of a similar set
of enzymes to those found in the Rhodophyceae.
We proposed recently that this complexity arose through the difficulty of readdressing a whole biochemical pathway from one cellular compartment (the cy‑
tosol) to another (the plastid) where it did not occur. Indeed, while re-addressing
an individual enzyme from one compartment to another can be simply achieved
and will be selected if it yields a benefit to an organism, re-addressing a whole bio‑
chemical pathway cannot be achieved simply in a single or restricted number of
steps. We have suggested that it was possible to reconstruct starch metabolism in
plastids by three successive stages involving the synthesis of first a small pool of
malto-oligosaccharides, then a larger pool of glycogen and, finally, the accumula‑
June 2009 © 2009 The Biochemical Society 3
Features
Metabolic pathways
Photosynthetic
CO2
fixation
Starch
CO2
Rubisco
x
H2
y
2H+
NAD(P)H
PQ (H2)
PS II
FNR
PGRL1?
Nda2
ATP
ADP + Pi
Fd
PGR5?
QA
NAD(P)H
H2ase
Cyt b6
f
PS I
Pc
2 H2O
O2 + 4H+
2H+
H+
Figure 2. Electron‑transfer pathways involved in hydrogen photoproduction in the unicellular
green alga C. reinhardtii i. Two electron‑transfer pathways of hydrogen production have been
described. The direct pathway (green arrows) involves PSII, the PQ pool, the cytochrome b6/f
complex and PSI. The indirect pathway (orange arrows) involves metabolic steps of starch breakdown, a NAD(P)H dehydrogenase (Nda2) involved in the non-photochemical reduction of the
PQ pool. In both pathways, reduced ferredoxin (Fd) supplies electrons to the Fe-hydrogenase
tion of starch5. In this process, all duplicated enzymes become subfunctionalized as
they had been optimized for the synthesis and degradation of these three distinct
substrates. Interestingly, the amount of subfunctionalization can be to some extent
predicted through the required sequence of events that, in turn, can be predicted
through our present knowledge of starch metabolism. This yields the precise distri‑
bution of duplications and subfunctionalization shown in the green algae and their
land plant derivatives5. Storage polysaccharide metabolism in algae thus demon‑
strates two different properties of biochemical pathways in these organisms: first,
a high degree of diversity is seen because of their polyphyletic nature (some algae
store β-glucans, whereas others store starch) and second, within a single group dis‑
tinct rewiring histories of merged pathways can yield vastly different outcomes, as is
the case for starch metabolism in the red and green algae.
Hydrogen photoproduction by microalgae
Because its combustion is clean and following technological improvements in fuel
cells, hydrogen is often considered as the energy carrier of the future. Unfortunately,
hydrogen is scarce on Earth. Therefore the development of a hydrogen economy
relies on our capacity to develop renewable and clean production technologies. As a
reflection of the great biochemical diversity outlined above, some microalgal species,
such as C. reinhardtii, harbour an unusual set of enzymes that enable them to ferment
starch during anoxia. Among these enzymes, hydrogenase resides within the chlo‑
roplast compartment and interacts with the photosynthetic electron‑transfer chain
(Figure 2). As a consequence, these organisms are able to produce hydrogen using
light as the sole energy source, hence the interest for hydrogen photoproduction7,8.
This phenomenon, which has been known since the 1940s from the pioneering work
of Gaffron and Rubin9, suffers from a major limitation related to the oxygen-sensi‑
tivity of the hydrogenase10. When anaerobically adapted cultures of C. reinhardtii are
illuminated, hydrogen production, which is highly efficient during the first minutes
of illumination (close to the maximal photosynthesis yield ~10%), rapidly stops be‑
4 June 2009 © 2009 The Biochemical Society
cause of the hydrogenase inhibition resulting from the
production of oxygen at Photosystem II (PSII). Note
that the selective advantage conferred by the existence
of a hydrogen photoproduction in oxygenic organ‑
isms is not clearly understood; it could be related to
the possibility of using the hydrogenase and hydrogen
production as a safety valve avoiding over-reduction of
photosynthetic electron carriers during the induction
of photosynthesis under anaerobic conditions. Melis
et al.11 proposed an experimental protocol based on
sulfur deficiency which also circumvents the oxygensensitivity of hydrogenase,i‑ resulting in a sustainable
hydrogen production. Sulfur deprivation triggers two
important phenomena at a cellular level, a rapid and
massive starch accumulation and a gradual PSII degra‑
dation12, resulting in a time-based separation between
an oxygenic phase of photosynthetic CO2 fixation, and
an anaerobic phase of hydrogen production. When the
rate of photosynthetic O2 evolution drops below the
rate of respiration, anaerobic conditions are reached
(provided that the microalgae are placed in a closed
photobioreactor), thereby triggering induction of hy‑
drogenase. Under these conditions, hydrogen can be
produced for several days using light alone11,13.
Starch metabolism and hydrogen
production
It was Gibbs and co-workers who first pointed out
the importance of starch fermentation in hydrogen
production14,15. By studying hydrogen production
in starchless C. reinhardtii mutants (sta6 and sta7),
Posewitz et al.16 proposed a central role for starch
metabolism in the hydrogen photoproduction proc‑
ess. Two different pathways can supply reductants
(as reduced ferredoxin) to the hydrogenase and so
sustain hydrogen production in light (Figure 2): a di‑
rect pathway involving PSII and the whole photosyn‑
thetic electron‑transfer chain, and an indirect pathway
which operated in the absence of PSII. The indirect
pathway relies on a non-photochemical reduction of
plastoquinones13,17. Starch catabolism was proposed to
play a role in both pathways13 (i) by sustaining mito‑
chondrial respiration and allowing the maintenance
of anaerobic conditions for the PSII-dependent direct
pathway, and (ii) by supplying electrons to the chlo‑
rorespiratory pathway and to the hydrogenase through
a Photosystem I (PSI)-dependent process during the
indirect pathway13,17,18. In the dark, fermentation in C.
reinhardtii is coupled to the degradation of starch re‑
serves, and a high rate of starch degradation generally
occurs in these conditions19. Transcript levels of two
β‑amylases presumably involved in starch degradation
markedly increased following dark anoxic acclima‑
Metabolic pathways
tion19. Whether corresponding enzymes or alternative
pathways are involved during the starch to hydrogen
conversion occurring in the indirect pathway during
light remains to be elucidated.
Towards the improvement of hydrogen
production
The introduction of experimental protocols based on
sulfur deficiency explains the considerable resurgence
of interest in hydrogen production by microalgae. Nu‑
trient starvation has, however, negative long‑term ef‑
fects on the efficiency of photosynthesis and therefore
on hydrogen production yields. One of the current
challenges towards improving hydrogen photopro‑
duction is to mimic the effects of sulfur deficiency,
i.e. the decrease of PSII activity and the accumulation
of starch, without relying on nutrient starvation. The
control of PSII activity using inducible promoters to
switch on or off the activity of PSII has recently been
proved to trigger hydrogen production efficiently20. A
major issue for future improvements will be to control
starch accumulation and breakdown without relying
on nutrient deficiency. This will require a thorough
understanding of signalling and regulatory pathways
involved in the sensing of nutrient status and in the
triggering of starch accumulation.
As mentioned above, maximal rates of hydrogen
production by the indirect pathway are lower than
by the direct pathway, indicating that metabolic steps
Features
specific to the indirect pathway, i.e. involving starch breakdown and/or reduction
of the PQ pool from stromal donors, limit the process21. If metabolic pathways of
starch biosynthesis are now rather well understood thanks to the characterization
of numerous starch‑less Chlamydomonas mutants22,23, little is known concerning the
metabolic pathways and regulations involved in starch breakdown in algae. Recent‑
ly, a type II NAD(P)H dehydrogenase (Nda2) involved in PQ reduction has been
demonstrated in C. reinhardtii chloroplasts24 and shown to be involved in hydrogen
production25. This enzyme, which reduces the PQ pool from stromal reductants,
probably participates in the indirect pathway of hydrogen production by supplying
electrons originating from starch breakdown to the intersystem electron‑transport
chain (Figure 2). This enzyme, and enzymes involved in starch breakdown (most
of which remain to be identified), may represent good targets for future biotechno‑
logical improvements.
■
Steven Ball attained an agronomy degree from the Faculty of Agronomy
in Gembloux (Belgium) and achieved his PhD in yeast molecular genetics
thanks to the research he conducted at the National Institutes of Health
(NIH) (Bethesda, MD). He secured a permanent position first as a research
assistant in Gembloux and then as a full professor of microbial genetics at
the University of Lille (France). He is leading a research team in Lille focused
on the study of starch metabolism in microalgae. email: [email protected]
Gilles Peltier studied microbiology and biochemistry at the University of Paris
7 and agronomy at AgroParisTech. He obtained a PhD in Biochemistry and
Plant Biology at the University of Aix-Marseille and conducted postdoctoral
research at the University of Georgia (Athens). He is currently leading a research
team at the CEA Cadarache (French Atomic Energy Commission), working on
mechanisms and regulations of light energy conversion by microalgae with
special interest in biofuel production. email: [email protected]
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June 2009 © 2009 The Biochemical Society 5