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in the case of RPs since in these cases, the biosynthetic machinery is the
ribosome. For the sunscreens, alkanes, alkenes and terpenes, the clus-
ters are small, and for geosmin, a single enzyme is responsible for the
biosynthesis. As illustrated in the following sections, the identification
of the biosynthetic clusters followed the same scenario in many cases:
the structure of the metabolites was known, feeding experiments using
isotopically labelled precursors gave some clues concerning the biosyn-
thesis (the incorporation of acetate reveals the involvement of PKSs, for
instance) and a particular gene was then identified by using degenerative
PCR amplifications. The link between the identified gene and the bio-
synthesis was then inferred from two different types of data: either the
genetic inactivation of the identified gene abolished the production of
the metabolite, an unambiguous and of course preferred experiment, or
a correlation was found in a series of producers and nonproducers of the
metabolite between the presence of the gene (genotype) and the produc-
tion of the metabolite (phenotype). This latter indirect evidence has been
used when genetic manipulation of the producer was not possible. Then,
using either genomic libraries cloned in cosmids or genome sequence
data, the cluster was identified and sequenced. In some interesting cases,
the gene clusters were directly identified by genome mining, for exam-
ple, in the case of alkane production (
Schirmer, Rude, Li, Popova, & del
Cardayre, 2010
).
As noted above, genetic manipulation of cyanobacteria is not always
possible and gene inactivation to prove the function of a specific cluster has
only been described in a few cases. In the other cases, in vitro characteriza-
tion of the biosynthetic enzymes afforded a firm experimental evidence
for the function of the identified cluster. But in some cases, there is still
no direct proof at all, except for the correlation between the biosynthetic
pathway predicted from the bioinformatic analysis of the gene cluster and
the biosynthesis imagined from the experimental data like isotopic feeding
experiments, or from the structure of the metabolite. In the case of small
cluster, like the cyanobactin gene clusters or the alkane gene cluster, heter-
ologous expression in
Escherichia coli
of the entire cluster afforded an elegant
proof for the function of the cluster. However, this type of experiment is
not always possible due to the size of the clusters.
In some cases, several clusters responsible for the biosynthesis of the
same metabolite have been sequenced, in different strains or genus, afford-
ing possible comparisons. These types of studies have been conducted in the
case of the
mcy
,
stx
,
cyr
,
aer, ana
and cyanobactin gene clusters (see section 4
