The anaerobic life of the photosynthetic alga Chlamydomonas reinhardtii Das anaerobe Leben der ...

141 Pages·2006·2.33 MB·English

The anaerobic life of the photosynthetic alga
Chlamydomonas reinhardtii
Photofermentation and hydrogen production upon
sulphur deprivation

Das anaerobe Leben der photosynthetischen
Alge Chlamydomonas reinhardtii
Photofermentation und Wasserstoffproduktion unter
Schwefelmangel

Dissertation zur Erlangung des Grades
eines Doktors der Naturwissenschaften
der Fakultät für Biologie
an der Internationalen Graduiertenschule Biowissenschaften
der Ruhr-Universität Bochum

angefertigt am Lehrstuhl für Biochemie der Pflanzen
in der Arbeitsgruppe Photobiotechnologie

vorgelegt von
Anja Christine Hemschemeier
aus Engelskirchen

Bochum
August 2005
Meinen Eltern und Meike.
In Liebe und Dankbarkeit.

„Wer sich nicht mehr wundern und in Ehrfurcht verlieren kann,
ist seelisch bereits tot.“

Albert Einstein
Teile dieser Arbeit wurden bereits veröffentlicht:

Happe T., Hemschemeier A., Winkler M. and Kaminski A. (2002) Hydrogenases in green
algae: Do they save the algae´s life and solve our energy problems? Trends Plant Sci 7,
246-250

Winkler M., Hemschemeier A., Gotor C., Melis A. and Happe T. (2002) [Fe]-
hydrogenases in green algae: Photo-fermentation and hydrogen evolution under sulfur
deprivation. Int J Hydrogen Energy 27, 1431-1439

Hemschemeier A. and Happe T. (2005) The exceptional photofermentative hydrogen
metabolism of the green alga Chlamydomonas reinhardtii. Biochem Soc Trans 33, 39-41

Fouchard S., Hemschemeier A., Caruana A., Pruvost J., Legrand J., Happe T., Peltier G.
and Cournac L. (2005) Autotrophic and mixotrophic hydrogen photoproduction in sulfur-
deprived Chlamydomonas cells. Appl Environ Microbiol, in press

Referent: Prof. Dr. Thomas Happe, Lehrstuhl für Biochemie der Pflanzen,
Arbeitsgruppe Photobiotechnologie
Koreferent: Prof. Dr. Franz Narberhaus, Lehrstuhl für Biologie der
Mikroorganismen

Tag der Abgabe: 26. August 2005
Diese Arbeit wurde gefördert durch die Studienstiftung des deutschen Volkes
Table of Contents
Table of contents 1
1 Introduction 1
1.1 Hydrogenases and hydrogen metabolism in unicellular green algae 1
1.2 Hydrogen production of sulphur-deprived C. reinhardtii cells 6
1.3 Fermentation: anaerobic energy production 11
1.4 The pyruvate formate-lyase system in E. coli 12
1.5 Objectives of this work 15
2 Materials and Methods 18
2.1 Organisms and growth conditions 18
2.1.1 Green algae 18
2.1.2 E. coli 19
2.2 Plasmids 20
2.3 Oligonucleotides 21
2.4 DNA and RNA techniques 21
2.5 Expression of C. reinhardtii pfl and pflA in E. coli 23
2.6 Western Blot Analyses 24
2.7 Physiological analyses of algal cultures 25
2.7.1 Quantification of hydrogenase activity 25
2.7.2 Measuring oxygen exchange with a Clark-type electrode 26
2.7.3 Detection of fermentative products and starch 26
2.7.4 Chlorophyll fluorescence measurements 27
2.7.5 Mass spectrometric analyses of the gas exchange in algal cultures 32
3 Results 36
3.1 Analysing the reasons leading to hydrogen production 36
3.1.1 Mass spectrometry as a tool for analysing gas exchange in C. reinhardtii 36
3.1.2 The analysis of C. reinhardtii mutant strains revealed three special phenotypes 40
3.1.3 A PSII-mutant offers clues to the electron source of hydrogen production 41
3.1.4 Is acetate essential for hydrogen production? 44
3.1.5 Hydrogen metabolism is delayed in a cytochrome oxidase deficient strain 46
3.1.6 A Rubisco-deficient strain produces hydrogen in the presence of sulphur 56
3.2 C. reinhardtii has an exceptional fermentative metabolism 63
Table of Contents
3.2.1 C. reinhardtii has several ethanol producing pathways 63
3.2.2 Fermentation is impaired in mutant strains FuD7 and CC-2803 65
3.2.3 The C. reinhardtii pfl and pflA cDNAs were isolated and characterised 66
3.2.4 C. reinhardtii PFL is functionally synthesised in E. coli 69
3.2.5 The algal PflA fails to activate E. coli PFL 73
3.2.6 Expression studies on selected genes 74
3.3 Overview of results 77
4 Discussion 78
4.1 What are the factors that finally lead to hydrogen production? 78
4.2 What kind of fermentative system is active in C. reinhardtii? 98
5 Summary 109
6 Zusammenfassung 111
7 References 113
8 Appendix 125
8.1. Assembly of the pflA-cDNA and deduced oligonucleotides 125
8.2. Alignments of PFL and PflA polypeptides 126
8.3. Annotated sequences encoding fermentative enzymes in C. reinhardtii 127
9 Curriculum vitae 129
Abbreviations
Abbreviations

aas amino acids
ACK acetate kinase
ADH Zn-containing alcohol dehydrogenase
ADP/ATP adenosine diphosphate / adenosine triphosphate
AOX alternative oxidase
bps base pairs
CDD Conserved Domain Database
cDNA “copy” desoxyribonucleic acid
CDS coding sequence
Chl chlorophyll
CoA coenzyme A
COX cytochrome oxidase
DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone
DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea
DNA desoxyribonucleic acid
EDTA ethylenediamine tetraacetic acid
FHL formate hydrogen lyase
FNR ferredoxin-NADP-oxidoreductase
GPD glycerol-3-phosphate dehydrogenase
GPP glycerol-3-phosphate phosphatase
HRP horse reddish peroxidase
HydA1 , 2 [Fe]-hydrogenases 1, 2 from C. reinhardtii
IPTG isopropyl-β-thiogalactoside
JGI Joint Genome Institute
KOG clusters of euKaryotic Orthologous Groups
LHC I, II light harvesting complex I, II
MOPS 3-(N-morpholinopropane)-sulfonic acid
mRNA messenger ribonucleic acid
NAD(P)H reduced nicotinamide adenine dinucleotide(phosphate)
NCBI National Center for Biotechnologial Information
Ndh1 rotenone-sensitive class I NAD(P)H dehydrogenase
Ndh2 rotenone-insensitive class II NAD(P)H dehydrogenase
PAGE polyacrylamide gel electrophoresis
PAM pulse amplitude modulating
PBS phosphate buffered saline
PCR polymerase chain reaction
PDC pyruvate decarboxylase
PetF algal ferredoxin
PFL pyruvate formate-lyase
PflA PFL activating enzyme, PFL activase
PFO pyruvate ferredoxin/flavodoxin-oxidoreductase
Abbreviations
PQ plastoquinone
PSI, PSII photosystem I, II
PTA1, 2 phosphotransacetylase 1, 2
PTOX plastidic terminal oxidase
Q , Q primary, secondary electron acceptor of P (PSII)
A B 680
RACE rapid amplification of cDNA ends
RNA ribonucleic acid
ROS reactive oxygen species
RT-PCR reverse transcriptase PCR
Rubisco ribulosebisphosphate-carboxylase/oxygenase
SAM S-adenosylmethionine
SDS sodium dodecylsulphate
SHAM salicyl hydroxamic acid
SQDG sulphoquinovosyl diacylglyceride
TAE Tris-acetate-EDTA
TAP Tris-acetate-phosphate
TdcE E. coli 2-ketobutyrate formate-lyase

Units of the International System of Units (SI) are not separately listed.

Gene designations

gene encoded protein
ack acetate kinase
adh1 alcohol- and acetaldehyde-dehydrogenase, PFL deactivase (Adh1/AdhE)
aox1, 2 alternative oxidase (AOX) 1, 2
atpB subunit of plastidic ATPase
coxI subunit COXI of mitochondrial cytochrome oxidase
cox90 subunit COX90 of mitochondrial cytochrome oxidase
hydA1, 2 [Fe]-hydrogenases HydA1, 2
pdc pyruvate decarboxylase
petA cytochrome f apoprotein of cytochrome b f complex
6
pfl pyruvate formate-lyase (PFL)
pflA PFL activating enzyme, PFL-activase
psbA core protein D1 of PSII
pta 1, 2 phosphotransacetylase (PTA) 1, 2
rbcL large subunit of Rubisco (RbcL)
sac1 regulator of specific responses to sulphur-deprivation (SAC1)
Introduction
1 Introduction
Research on the unicellular chlorophyte alga Chlamydomonas reinhardtii (fig 1-1) is
almost one century old now, and this organism, sometimes called the “photosynthetic
yeast” (Rochaix, 1995), has become an important model (Harris, 2001). A simple life cycle
that can be easily manipulated, rapid growth and a haploid genome in vegetative cells are
characteristics that make this alga an ideal model system. Although the Volvocales are
considered to be a side-branch of the phylogenetic tree leading to higher land plants
(Chapman and Buchheim, 1992), the photosynthetic apparatus is nevertheless highly
conserved, making C. reinhardtii an excellent model system for the elucidation of
photosynthesis in vascular plants. Due to its ability to grow heterotrophically on acetate,
C. reinhardtii is useful for the study of photosynthesis and chloroplast biogenesis, since
photosynthetic mutants are viable on acetate (Harris, 1989). C. reinhardtii, however, also
has its very own mysteries, since it possesses features that are quite unusual for a
eukaryotic photosynthetic organism. This alga differs from other eukaryotes by having a
complex fermentative metabolism that is
10 µm
marked by the production of hydrogen gas and
formate.

Fig 1-1: Light microscopic photograph of the
unicellular green alga C. reinhardtii (A. Kaminski,
Rheinische Friedrich-Wilhelms-Universität Bonn).

1.1 Hydrogenases and hydrogen metabolism in unicellular green algae
In the first half of the last century it was observed that the unicellular green alga
Scenedesmus obliquus, after adaptation to anaerobic conditions, develops a hydrogen
metabolism. It can use the reductive power of hydrogen for the assimilation of carbon
dioxide (Gaffron, 1939), and it is also able to photoproduce hydrogen (Gaffron and Rubin,
1942). Other investigators observed photosynthetic hydrogen evolution in anaerobic
cultures of Chlorella fusca (Spruit, 1958) and Chlamydomonas moewusii (Frenkel, 1952).
In 1974, it was concluded that many species of unicellular green algae have a hydrogen
metabolism (Kessler, 1974). One of the most efficient hydrogen producers was
1
Introduction
C. reinhardtii (Stuart and Gaffron 1972; Ben-Amotz et al., 1975). Its hydrogenase enzyme,
HydA1, was purified to homogeneity and biochemically characterised in the 1990s (Happe
and Naber, 1993). It turned out to be a small iron-containing protein of 48 kDa, which is
localised in the chloroplast stroma. In all likelihood, the photosynthetic ferredoxin PetF is
the natural electron donor to the hydrogenase (Happe et al., 1994). HydA1 is very sensitive
to molecular oxygen and only synthesised under anaerobic conditions. Hydrogenase
activity is detectable very soon (~ 5 min) after oxygen has been removed from a
C. reinhardtii culture and Northern blot analyses indicated transcriptional regulation of the
hydA1 gene in response to anaerobiosis (Happe and Kaminski, 2002). More recent research
using reporter gene assays confirmed these findings and showed that the hydA1 promoter is
regulated in response to levels of oxygen within the cells (Stirnberg and Happe, 2004).
Indeed, isolation of the hydA1 gene, which had proved unsuccessful until recently, was
achieved by making use of the anaerobic expression of hydA1. Applying the method of
suppression-substractive hybridisation PCR, which is a tool to identify differentially
expressed genes, a cDNA sequence encoding the [Fe]-hydrogenase of C. reinhardtii could
be identified in total mRNA isolated from an anaerobic algal culture (Happe and Kaminski,
2002). The deduced amino acid sequence of HydA1 revealed the highly conserved
C-terminal domain characteristic for [Fe]-hydrogenases, including four cysteine residues
that coordinate the active site, the so-called H-cluster (Peters et al., 1998).

Based on these fundamental data, the hydA genes of further algal species were isolated in
the following years (Florin et al., 2001; Winkler et al., 2002a; Winkler M., personal
communication). It turned out that the hydrogenase enzymes of unicellular green algae
represent a novel type of [Fe]-hydrogenases (Happe et al., 2002). Chlorophycean type
[Fe]-hydrogenases are small proteins (44 to 48 kDa) and lack the N-terminal ferredoxin-
like domain present in all other [Fe]-hydrogenases isolated to date (Vignais et al., 2001).
This so-called F-domain harbours two or more additional Fe-S-clusters which are thought
to be involved in the transfer of electrons between the external electron donor / acceptor
and the catalytic centre (Peters et al., 1998; Nicolet et al., 1999). The absence of any
additional redox clusters in the hydrogenases of green algae indicates a novel electron
transfer pathway for these enzymes and suggests direct electron transfer between the
electron donor and the H-cluster of HydA1. Protein modelling studies indicate a single
positively charged binding site in an otherwise negatively charged molecule. A polypeptide
stretch in the C-terminal part of the primary structure, which is unique for algal
2