The violacein biosynthesis monitored by multi-wavelength fluorescence spectroscopy and by the PARAFAC method

Article abstract of DOI:10.1590/S0103-50532012005000083

Obtaining information about a biosynthetic pathway is a complex and laborious procedure. In this sense, this work presents a new approach for the initial analysis of the biosynthesis of fluorescent natural products using as example the violacein biosynthe

Full text of DOI:10.1590/S0103-50532012005000083

J. Braz. Chem. Soc., Vol. 23, No. 11, 2054-2064, 2012.
Printed in Brazil - ©2012 Sociedade Brasileira de Química
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Article
A
The Violacein Biosynthesis Monitored by Multi-Wavelength
Fluorescence Spectroscopy and by the PARAFAC Method
Clecio Dantas, Pedro L. O. Volpe, Nelson Durán and Márcia M. C. Ferreira*
Institute of Chemistry, University of Campinas (UNICAMP), CP 6154, 13083-970 Campinas-SP, Brazil
A obtenção de informações sobre uma rota biossintética é um procedimento complexo e
trabalhoso. Neste sentido, o presente trabalho apresenta uma nova abordagem para a análise inicial
da biossíntese de produtos naturais fluorescentes usando a biossíntese da violaceína como exemplo.
Para tanto, uma cultura da Chromobacterium violaceum foi inoculada em um biorreator de onde
alíquotas eram retiradas a cada 2 h para posterior análise por espectroscopia de fluorescência 2D.
As matrizes de emissão-excitação obtidas demonstraram o comportamento dinâmico dos sinais
de fluoróforos que são consumidos e produzidos pela bactéria. Estes sinais foram resolvidos pelo
método PARAFAC (análise paralela de fatores) perfazendo um total de seis espécies químicas.
O triptofano e a violaceína foram identificados por comparação espectral. A identificação dos
outros fluoróforos mostrou-se a etapa crítica devido à falta de banco de dados de produtos naturais
fluorescentes para comparação. Por fim, esta metodologia apresenta um grande potencial para
fornecer informações da biossíntese de produtos naturais.
Obtaining information about a biosynthetic pathway is a complex and laborious procedure.
In this sense, this work presents a new approach for the initial analysis of the biosynthesis of
fluorescent natural products using as example the violacein biosynthesis. For this, a culture of
Chromobacterium violaceum was grown in a bioreactor from which aliquots were collected every
2
h for subsequent analysis by multi-wavelength fluorescence spectroscopy. The excitation-emission
matrices demonstrated the dynamic behavior of the fluorophores signal that are consumed and
produced by the bacterium. These signals were resolved by PARAFAC (parallel factor analysis)
method totalizing six pure components. Tryptophan and violacein were identified by comparison
to spectra available in the literature. The identification of other fluorophores was critical step due to
the lack of a database of fluorescent natural products to compare spectra. Finally, this methodology
has great potential to achieve a deeper insight into the biosynthesis of natural products.
Keywords: biotransformation, violacein, fluorescence spectroscopy, curve resolution
Introduction
Natural products have had historical success as
biologically active structures. Among these, the violet
pigment, named violacein, produced mainly by bacteria
of the genus Chromobacterium has attracted increased
interest owing to its important biological activities and
1,2
pharmacological and industrial potentials.
Violacein(Figure1)isasecondarymetabolitewithamolar
mass of 343.3 amu and is constituted of 5-hydroxyindole,
2
-pyrrolidone and 2-oxindole moieties that show strong
Figure 1. Chemical structure of violacein.
3
absorption in the visible region due to resonance.
Studies about violacein biosynthesis began in 1934
with Tobie, who observed that oxygenation of a
Chromobacterium violaceum culture greatly reduced the
4
time required for maximum pigment production. Twenty
5
,6
five years later, DeMoss and Evans discovered that
to synthesize violacein, the bacteria needed molecular
*
e-mail: marcia@iqm.unicamp.br

Vol. 23, No. 11, 2012
Dantas et al.
2055
oxygen and that L-tryptophan was incorporated into
violacein, except for the carboxylic carbon, eliminated by
a decarboxylation process during the biosynthesis. From
fusion of two tryptophan-derived units. Additionally,
the authors found a level of identity of 34% between
VioB and RebD (a heme protein required for formation of
CPA and involved in the biosynthesis of the indocarbazoles
rebeccamycin and staurosporine). Based on the similarities
7-9
1987 to 1990, Hoshino et al. demonstrated that (i) the
carbon skeleton of the pyrrolidone moiety was built up by
the condensation of the side chains of two L-tryptophan
molecules accompanied by the 1,2-shift of the indole ring
1
7
between these two pathways, Sánchez et al., in 2006,
hypothesized the construction of hybrid pathways for
production of oxygenated indolocarbazole, by coexpression
of the genes. The hybrid pathways did not yield oxygenated
indolocarbazoles, however their results provided new
information on violacein biosynthesis. First, a pair of genes
(VioAB), responsible for the earliest steps in violacein
biosynthesis, was equivalent to the homologous pair in the
indolocarbazole pathway (RebOD), directing the formation
of CPA. Second, in addition to VioABCD, a fifth gene
(VioE) was essential for violacein biosynthesis. Third,
CPA was not an intermediate product of this biosynthesis.
However, its formation was not independent of violacein
biosynthesis as it was produced by VioAB in the absence
ofVioE. Fourth, bothVioC andVioD oxygenases appeared
to act on the latter steps of violacein biosynthesis. The
last two results were contradictory to those reported by
7
on one molecule of tryptophan; (ii) all carbon, nitrogen and
hydrogen atoms in the pyrrolidone moiety were provided
exclusively by L-tryptophan and that the oxygen atoms
8
come from molecular oxygen; and (iii) the intermediacy
of 5-hydroxy-L-tryptophan in the violacein biosynthesis
since the hydroxylation of tryptophan was the first step of
9
violacein biosynthesis.
After that, the violacein biosynthesis has only been
evaluated by radiolabel incorporation experiments. In 1991,
10
Pemberton et al. began a study at the genetic level by
isolating a 14.5 kb fragment containing the genes encoding
violacein biosynthesis from C. violaceum. From 1993 to
11-14
2000, in a series of works, Hoshino et al.
discovered that
(
i) chromopyrrolic acid (CPA) was produced independently
1
1
from the violacein biosynthesis; (ii) proviolacein,
prodeoxyviolacein and pseudoviolacein were identified
as plausible biosynthetic intermediates of violacein and
that oxygenation the 2-position of the indole ring occurs
9
,11,14
Hoshino et al.,
and, consequently, to the mechanism
15
proposed by August et al.
1
8
In 2006, Balibar and Walsh reconstituted in vitro the
entire violacein pathway. Their results demonstrated that:
(i) L-tryptophan, and not 5-hydroxy-L-tryptophan, was
the sole precursor of violacein; (ii) VioB was responsible
for the oxidative coupling of two molecules of IPA
imine generated by VioA for formation of the pyrrole
core; (iii) the fifth gene, VioE, was responsible for the
1,2-shift of the indole ring that resulted in the formation
of prodeoxyviolacein rather than CPA; (iv) formation
of violacein then proceeded by the sequential action of
VioD andVioC. These results confirmed the in vivo results
1
2
in a final step in this biosynthesis; (iii) oxygenases
were involved and cofactor NADPH was needed for the
13
biosynthesis of violacein; and (iv) that the 1,2-shift of
the indole ring occurred through an intramolecular process
during the formation of the left part (5-hydroxyindole side)
14
of the violacein skeleton.
1
5
In 2000, August et al. showed that the violacein
biosynthetic gene cluster was comprised of four genes,
VioABCD. The products of the VioA, VioC and VioD
were nucleotide-dependent monooxygenases. Disruption
of VioA or VioB completely abrogated the biosynthesis
of violacein intermediates, while disruption of VioC
or VioD genes resulted in the production of violacein
precursors. Collecting all information obtained so far,
1
7
proposed by Sánchez et al.
1
9
In 2007, Shinoda et al. reported the identification
of a true intermediate of violacein biosynthesis, named
protoviolaceinic acid, which was produced by incubating
L-tryptophan with VioABDE in the presence of NADPH.
The production of this compound confirmed that VioC
works to oxygenate the 2-position of the right side
indole ring. Additionally, their results indicated that the
oxygenation reaction to form the central pyrrolidone core
proceeded in a non-enzymatic fashion. An overview of the
violacein biosynthesis proposed by these authors is shown
in Figure 2.
1
5
August et al. proposed a hypothetical route for violacein
biosynthesis from L-tryptophan. This pathway consisted
of: (i) modification of one L-tryptophan by VioD, forming
5
-hydroxy-L-tryptophan, and oxidative deamination of
the other by VioA, producing indole-3-pyruvic acid (IPA);
ii) decarboxylative fusion of these two tryptophan-derived
(
units and a 1,2-shift of the 5-hydroxyindole ring by VioB,
resulting in prodeoxyviolacein; and (iii) oxygenation of the
latter by VioC yielding violacein.
It begins with the VioA oxidation of L-tryptophan to
indole-3-pyruvic acid (IPA) imine. Then, the oxidative
coupling of two molecules of IPA imine by VioB gives
an uncharacterized intermediate proposed to be an IPA
16
In 2005, Howard-Jones andWalsh reported a similarity
between violacein biosynthesis and indolecarbazole
biosynthesis. Both pathways include a decarboxylative

2
056 The Violacein Biosynthesis Monitored by Multi-Wavelength Fluorescence Spectroscopy and by the PARAFAC Method
J. Braz. Chem. Soc.
structural elucidation, molecular tools and recombinant
methods were used. However, a special property of this
system was not explored. In the violacein biosynthesis,
the precursor (L-tryptophan), the product (violacein) and,
probably, the intermediaries are fluorescent compounds.
Hence, it is expected that information about this biosynthesis
can be obtained from fluorescence spectroscopy when it
is applied to detect compounds that are consumed and
produced during cultivation.
Thus, a methodology for obtaining information about
the biosynthetic pathway of fluorescent compounds is
proposed in this work. It consists in employing multi-
wavelength fluorescence spectroscopy and PARAFAC
method to detect and identify (through spectral resolution)
fluorophores that are consumed and produced by a
bacterium and, finally, to establish a relationship with the
natural product biosynthesis.
The combination of the multi-wavelength fluorescence
spectroscopy and the PARAFAC method has already been
applied to monitor bioprocesses in which the time evolution
profiles obtained for the fluorophores are related to the
20-23
target bioprocess variables.
However, this methodology
was not employed to directly link the spectral resolution
information to a biosynthetic pathway. Therefore, this is a
new and complementary approach for the initial analysis
of the biosynthesis of fluorescent natural products.
Experimental
Organisms, culture media and cultivation conditions
Starting from a culture of C. violaceum CCT 3496,
a colony was isolated, after culture by the streak plate
method. This pure colony was inoculated into a 250 mL
Erlenmeyer flask containing 50 mL of sterilized culture
medium (0.50% D-glucose, 0.50% bacteriologic peptone,
0.25% yeast extract and 0.03% L-tryptophan) and grown for
24 h at 33 ºC on an orbital shaker at 200 rpm. Afterwards,
10 mL of this bacterial culture was transferred to a 1500 mL
BioFlo bioreactor (New Brunswick Scientific) containing
1000 mL of sterilized culture medium and the parameters
temperature, stirring and air flow were adjusted (33 ºC,
Figure 2. Overall pathway to violacein biosynthesis.
imine dimer. The skeleton of this compound is catalytically
arranged byVioE through an intramolecular rearrangement
of the indole ring producing protodeoxyviolaceinic acid.
This latter can be converted spontaneously by autoxidation
into prodeoxyviolacein or it can undergo hydroxylation
catalyzed by VioD at the 5-position of the left side indole
ring giving protoviolaceinic acid. The protoviolaceinic
acid is hydroxylated by VioC at the 2-position of the right
side indole ring to form violaceinic acid. The subsequent
conversion to violacein involves a non-enzymatic process
of oxidative decarboxylation.
-1
200 rpm and 1.0 L min , respectively) and kept constant
throughout the cultivation (for 36 h).
Sample collection
The mechanistic investigation of this complex
biosynthetic pathway at the chemical, biochemical and
genetic levels involved many steps. Radiolabel incorporation
experiments coupled with techniques for separation and
Every 2 h, aliquots of 10 mL were withdrawn from the
bioreactor with the aid of a glass syringe, stored in 15 mL
Falcon tubes and frozen (-20 ºC). At a later stage, each
aliquot was thawed in a thermostatic bath at 30 ºC for

Vol. 23, No. 11, 2012
Dantas et al.
2057
5
min and then centrifuged at 7000 rpm for 10 min. The
problem, new instrumental settings were tested. Filters
were set on “Open” for both excitation and emission
monochromators, which means that no filter was used and
the photomultiplier tube (PMT) voltage was increased to
800 V. Usually, excitation and emission filters are used to
reduce the stray radiation effect. Additionally, the output
signal for a given amount of light will be enhanced by
increasing the PMT voltage.
supernatant was eliminated and 5 mL absolute ethanol was
added to extract intracellular compounds, which were then
centrifuged at 7000 rpm for 10 min for cell removal and the
new supernatant was collected. The crude ethanolic extract
was passed through a 0.45 μm pore-size filter. Altogether,
18 samples corresponding to the crude ethanolic extract
of C. violaceum at different stages of biosynthesis were
collected.
These new instrumental settings allowed visualization
of fluorescence signals in the low energy region as shown
in Figure 3b. Hence, it was decided to investigate the entire
spectral range by dividing it into smaller regions and using
specific instrumental settings for each. Altogether, five
regions were defined (Figure 3a) and their instrumental
settings are summarized in Table 1.
Following this procedure, six EEMs, covering the entire
spectral region and the regions B, C, D, E and F were
collected for each sample.
Multi-wavelength fluorescence spectroscopy
Fluorescence measurements were performed using a
Varian Cary Eclipse fluorescence spectrophotometer in
the scan mode. Aliquots of 0.5 mL of the samples were
diluted in 2.5 mL of absolute ethanol in the quartz cuvette to
reduce effects of quenching, inner filter and energy transfer
processes. Initially, the excitation-emission matrices
(
EEMs) were collected for each sample with spectral
ranges of 250-620 nm for excitation and 270-800 nm for
Data preprocessing
-1
emission. The scan rate for each EEM was 1200 nm min .
The increments for the excitation and emission wavelengths
were 5 and 2 nm, respectively. The excitation and emission
slits were adjusted to 5 nm. Filters were set on “Auto”
for both excitation and emission monochromators.
This means that the filter wheel is automatically moved
to the appropriate position according to the selected
excitation/emission wavelength. The photomultiplier tube
Rayleigh scatter peaks are largely unrelated to the
chemical properties of the sample. They occur when a
molecule has been excited to a virtual energy state by
a photon with insufficient energy to completely excite
2
4
the molecule. They are situated diagonally in EEMs
(Figure 3a) and do not present linear behavior which may
complicate the fluorescence data modeling. Therefore,
Rayleigh scatter peaks should be treated prior to modeling.
(PMT) voltage was set to 600 V. The measurements were
2
5-28
carried out in 90° geometry.
Many approaches have been developed for that.
It is known that the violacein molecule shows an
emission band at 675 nm when excited at 575 nm.
However, with the measurement conditions described
above, it was not possible to detect fluorescence signals
in the spectral region of 550-620 nm for excitation and
The procedure employed here consisted in removing and
replacing affected areas by interpolated values according
to the data in the remaining parts of EEM. Interpolation
of scattered areas was performed by applying the method
2
2
9
developed by Bahram et al., which is available from
30
6
00-800 nm for emission (Figure 3a). To explore this
another work.
Figure 3. (a) 2D contour plot of an EEM and spectral range of each region; (b) 3D surface and contour plots of an EEM in the spectral region of 550-620 nm
for excitation and 600-800 nm for emission.

2
058 The Violacein Biosynthesis Monitored by Multi-Wavelength Fluorescence Spectroscopy and by the PARAFAC Method
J. Braz. Chem. Soc.
Table 1. Measurement conditions for each spectral region
Excitation
range / nm
Emission
range / nm
Excitation
filter
Emission
filter
PMT
voltage / V
Excitation
slit / nm
Emission
slit / nm
Region
B
C
D
E
F
250-320
325-395
400-470
475-545
550-620
300-600
375-675
450-750
525-800
600-800
auto
auto
open
open
open
auto
auto
open
open
open
600
600
800
800
800
5
5
5
5
5
5
5
5
5
5
PMT: photomultiplier tube.
Data modeling
The trilinear model is found to minimize the sum of
squares of the residuals, e , according to equation 1,
ijk
Multi-wavelength fluorescence spectroscopy involves
successive acquisition of emission spectra at multiple
excitation wavelengths generating a two-dimensional
matrix (EEM) per sample. Using the culture time, it is
possible to produce a 3D array X with dimensions I × J × K
by stacking EEMs one on top of another (Figure 4), in
which I is the number of samples collected during the
reaction, J is the number of emission wavelengths and K
is the number of excitation wavelengths.
(1)
th
th
where, x is the intensity of the i sample at the j emission
ijk
th
wavelength and at the k excitation wavelength. The a
if
element may be interpreted as the relative concentration
of analyte f in sample i. The j-vector b with elements b
f
jf
(j = 1,…, J) is the estimated emission spectrum of this
analyte and likewise c is the estimated excitation spectrum.
The most important step when applying the PARAFAC
method is to estimate the appropriate number of components
or factors (F) in the data set (see equation 1). This number
can be determined based on several criteria as, for example,
f
3
1
This array X can be modeled by PARAFAC,
a
trilinear decomposition method for higher order data
whose structural basis is given by three loading matrices A,
B and C (Figure 4). These matrices are obtained using an
alternating least squares algorithm and contain information
related to relative concentration, emission spectrum and
excitation spectrum of each analyte. The core H is a binary
array (F × F × F) with ones in the super-diagonal and
zeros in all other elements and indicates that only loading
vectors with the same column number interact, i.e., only
2
visual appearance, explained variance (R ), core consistency
32-34
diagnostic (CORCONDIA), among others. In this work,
the number of components was assessed on the basis of
three criteria: explained variance, CORCONDIA and lack
of fit (LOF) of the models.
The explained variance (R ) is calculated by taking into
account the sum of the squares of the residuals, e , and
2
th
th
the f column of A interacts with the f columns of the
B and C matrices.
ijk
the sum of the squares of the elements of the array X, xijk,
The fluorescence excitation-emission matrices are
known to approximately follow a trilinear model.
according to equation 2.
3
2
Therefore, the application of the PARAFAC method
in fluorescence excitation-emission data can resolve
overlapping signals into pure concentration and spectral
profiles.
(2)
Figure 4. Three-way data array obtained by stacking EEMs for each sample and graphical representation of PARAFAC decomposition.

Vol. 23, No. 11, 2012
Dantas et al.
2059
CORCONDIA is defined as
(3)
where, gdef is the calculated element of the core using the
PARAFAC model; hdef the element of a binary array and F
is the number of factors in the model. In an ideal PARAFAC
model, gdef is equal to hdef and, in this case, CORCONDIA
will be equal to 100%.A model with a CORCONDIA value
above 90% can be understood as “very trilinear”, whereas
a model with a CORCONDIA in the neighborhood of
Figure 5. Visual changes occurring during the violacein biosynthesis.
accumulation of toxic products. It is in the stationary phase
that several secondary metabolites (among them, violacein)
50% would mean a problematic model with signs of both
38
trilinear and not trilinear variations.A CORCONDIA close
to zero or even negative implies an invalid model. More
are synthesized.
33
detailed information can be seen in elsewhere.
Fluorescence data
In complex biological matrices, the determination of
the number of factors by CORCONDIA is sometimes
elusive and cannot be automated. In this sense, the
appropriate number of factors should be evaluated by
other criteria. Here, the LOF criterion is introduced and
calculated according to equation 4.
The fluorescence data set consisted of 18
excitation-emission matrices (EEMs) of ethanolic extracts
of C. violaceum collected at different stages of violacein
biosynthesis. The fluorescence landscapes were initially
measured at 266 emission wavelengths (from 270 to 800 nm
with 2 nm intervals) and 75 excitation wavelengths (from
3
5
2
50 to 620 nm with 5 nm intervals) providing a detailed
map of the fluorescence properties of the sample. The
dynamic behavior of fluorophores involved in the violacein
biosynthesis is shown in Figure 6 in the form of contour
plots of some preprocessed EEMs.
(4)
In Figure 6a, the first fluorophore shows a broad band
of emission between 330 and 370 nm when excited from
270-290 nm. This compound seems to be consumed
during the fermentation since the intensity of the emission
band decreases with the reaction time. The second
fluorophore shows two excitation maxima (270-285 and
The PARAFAC algorithm used in this work is from
the N-way Toolbox, downloaded from elsewhere and
used with the software MatLab 7.0 (Mathworks Inc, USA).
36
37
Results and Discussion
2
95-320 nm) and one emission maximum between
Growth phases of bacteria
400-450 nm. The third fluorophore shows three excitation
maxima (315-320, 325-340 and 345-355 nm) and one
emission maximum from 440-480 nm. These last two
compounds are possible intermediates in the violacein
biosynthesis since they are being produced with the
progress of the fermentation.
The violacein biosynthesis was monitored in the
bioreactor for 36 h producing a total of 18 samples. The
visual changes that occurred in the culture medium during
this period are presented in Figure 5.
The first hours after inoculation corresponded to a lag
phase that is the period of adaptation of bacteria to the
environment and is characterized by intense metabolic
activity and the absence of cell division. Following the
lag phase is the exponential or log phase, in which the
population grows in a logarithmic fashion. This phase can
be easily visualized by the turbidity of the culture medium.
Finally, it is possible to see the stationary phase where the
growth rate slows down as a result of nutrient depletion and
As previously described in the Experimental section,
with the initial instrumental settings it was not possible to
detect the fluorescence signal from violacein. However,
the violet color of the culture medium (Figure 5) indicates
that this compound is being biosynthesized, a fact that was
confirmed by UV-Vis absorption spectroscopy (data not
shown). This apparent contradiction is probably caused by
the dependence of sensitivity of fluorescence in both the
fluorophore and the instrument.

2
060 The Violacein Biosynthesis Monitored by Multi-Wavelength Fluorescence Spectroscopy and by the PARAFAC Method
J. Braz. Chem. Soc.
Figure 6. Kinetic evolution of EEMs obtained during the violacein biosynthesis (a) entire spectral range and (b) low energy regions (D, E and F).
To assess this problem, the entire spectral range was
divided into five smaller regions, defined in Table 1, and
specific measurement conditions were used for each of
them. This procedure produced 18 fluorescence landscapes
for each region.
The dynamic behavior of fluorophores in the low energy
regions (D, E and F regions) is shown in Figure 6b, in which
new EEMs were created by juxtaposition of preprocessed
D, E and F EEMs to give an overview of the fluorescence
signals. The missing values were set to NaN.
The fluorophore with maximum emission between
590-620 nm shows three excitation maxima (450-470,
525-535 and 550-570 nm). In this case, the absorption
peak of lowest energy is due to fluorophore excitation to
an accessible vibrational level of the first electronic excited
state of the same multiplicity. The absorption peak of

Vol. 23, No. 11, 2012
Dantas et al.
2061
highest energy is probably due to fluorophore excitation
to an accessible vibrational level of the second electronic
excited state of the same multiplicity. The only emission band
observed indicates the occurrence of the internal conversion
process, wherein excited molecules generally relax toward
the lowest vibrational level within the first electronic
excited state. Also it is possible to observe a fluorophore
that is produced during the fermentation and presents broad
excitation (400-460 nm) and emission (450-540 nm) bands.
Analyzing the results in Table 2 for the entire spectral
range and for region B, it can be inferred that the explained
variance and LOF values indicate that a model with
three components is suitable since the improvement
obtained in these parameters when one more component
is included in the model is very small (0.12 and 0.98
for entire spectral range and 0.13 and 1.34 for region B,
respectively). On the other hand, the CORCONDIA values
obtained from PARAFAC calculations upon varying the
number of components for these two regions show that
the optimal number of components is not very clear
PARAFAC modeling
(
a three component model has CORCONDIA values
Pure excitation and emission profiles for each of the
six regions were extracted from the preprocessed EEMs
by applying PARAFAC. The appropriate number of
components in each data set was determined based on the
explained variance, CORCONDIA and LOF values.
PARAFAC models with non-negativity constraints
in the three modes were calculated from one to six
components and the relative values of explained variance,
CORCONDIA and LOF are listed in Table 2.
of 99.58 and 99.01% for the entire spectral range and
for region B, respectively, while a four component has
CORCONDIA values of 70.68 and 72.69%). However,
the graphical visualization of spectral loading matrices
obtained for PARAFAC models with four components
for the entire spectral range and for region B shows that,
except for the fourth species, the shape and position of
the recovered profiles do not change. This feature is
consistent with the trilinearity property. In this way, it is
Table 2. Percentage of explained variance, CORCONDIA and LOF, calculated for trilinear models using the PARAFAC method with 1 to 6 components
No. components
1
2
3
4
5
6
Entire spectral range
Explained variance / %
CORCONDIA / %
LOF / %
82.30
100
96.03
99.93
19.93
99.56
99.68
70.65
5.63
99.75
11.58
5.02
99.83
5.69
4.08
99.58
6.61
42.07
Region B
Region C
Region D
Region E
Region F
Explained variance / %
CORCONDIA / %
LOF / %
80.85
100
98.35
99.97
12.85
99.70
99.01
5.49
99.83
72.69
4.15
99.87
25.83
3.62
99.93
13.31
2.70
43.76
Explained variance / %
CORCONDIA / %
LOF / %
98.91
100
99.71
99.93
5.38
99.89
22.65
3.34
99.95
11.25
2.20
99.96
-0.23
1.90
99.97
-0.05
1.65
10.45
Explained variance / %
CORCONDIA / %
LOF / %
75.03
100
96.51
99.77
18.69
98.16
51.39
13.55
98.83
33.75
10.82
99.21
12.41
8.87
99.63
4.07
6.09
49.97
Explained variance / %
CORCONDIA / %
LOF / %
86.65
100
97.13
99.98
16.94
97.96
35.39
14.26
98.50
42.35
12.23
98.87
7.64
99.14
6.14
9.26
36.53
10.62
Explained variance / %
CORCONDIA / %
96.18
100
98.60
97.72
11.82
99.44
55.56
7.49
99.63
27.05
6.09
99.67
11.73
5.57
99.71
-1.21
5.36
LOF / %
19.55
2
R : explained variance; CORCONDIA: core consistency diagnostic; LOF: lack of fit.

2
062 The Violacein Biosynthesis Monitored by Multi-Wavelength Fluorescence Spectroscopy and by the PARAFAC Method
J. Braz. Chem. Soc.
concluded that the fourth component is not a chemical but
a physical component. Therefore, the number of factors
was chosen to be three for both regions. This observation
reinforces that the determination of the number of factors
by CORCONDIA in complex biological matrices is
sometimes elusive and cannot be automated.
biosynthesis. The second fluorophore has an emission peak
at 423 nm when excited at 280 and 308 nm (see Figure 6a,
sample-20 h). This compound is probably a tryptophan
derivative since the excitation at 280 nm is due to the
indole nucleus. The double absorption can be understood
as electronic transitions to different excited states or as
electronic transitions between two different vibrational
levels in the same excited state. The concentration profile
indicates that this fluorophore reaches its maximum
concentration after approximately 30 h and then begins to
be consumed. Unfortunately, it was not possible to identify
this compound by comparison to spectra available in the
literature. However, the concentration profile suggests that
it is an intermediate of the violacein biosynthesis. The third
fluorophore shows three excitation peaks at 315, 330 and
347 nm and one emission peak at 470 nm (see Figure 6a,
sample-36 h). The concentration profile indicates that
this fluorophore starts to be produced after approximately
18 h and reaches its maximum concentration at 32 h.
The emission profiles obtained by the resolution in
the regions B and C are the same visualized in Figure 7a.
The excitation profiles are also the same. However, as
the excitation range is smaller, the excitation profiles are
truncated. The juxtaposition of the excitation profiles
obtained for the regions B and C recovers the excitation
profile of the entire spectral range.
For regions C and E, the explained variance, LOF
values and CORCONDIA values indicate that two is an
appropriate number of factors to model the data. Although
these same criteria point to three factor models for regions
D and F, it was assumed that two is a more reasonable
value. This choice was based on drastic variation of the
CORCONDIA values for models with 3 or more factors
39
when N-way Toolbox and PLS-Toolbox were run several
times (an indication of ill-posed models).
The concentration, emission and excitation profiles
obtained by the PARAFAC models for each region, with
the suitable number of components, are shown in Figure 7.
In all, six chemical species probably involved in
the biosynthesis of violacein could be detected by this
methodology. In Figure 7a, the first excitation profile has a
narrow maximum centered at 280 nm and a sharp emission
maximum at 342 nm. The respective concentration profile
shows a maximum concentration at 6 h (lag phase) and
then decreases with reaction time. The emission loadings
40
are similar to the pure emission spectrum of tryptophan.
This fact corroborates the mechanism presented in Figure 2,
in which tryptophan is the precursor in the violacein
The fluorophore represented by the continuous red line
in Figures 7b shows similar concentration profiles to that of
Figure 7. Estimates of emission and excitation spectra obtained by PARAFAC: (a) entire spectral range and (b) regions D, E and F juxtaposed.

Vol. 23, No. 11, 2012
Dantas et al.
2063
tryptophan. This compound seems to have three excitation
peaks at 460, 525 and 560 nm and one emission peak close
to 605 nm (see Figure 6b, sample-8 h).
Another important conclusion is to show that the use
of different instrumental settings is important to enable the
visualization of fluorescence signals in the lower energy
spectral region due the wavelength dependent sensitivity
of the spectrofluorometer PMT.
4
1
Asamizu et al. reported the electronic absorption
spectra of StaD, an enzyme involved in saturosporine
biosynthesis that is a homolog to VioB and responsible
for the coupling reaction between two molecules of
Finally, this methodology has great potential to achieve
a deeper insight into the biosynthesis of natural products.
+
indole-3-pyruvic acid and NH4 to yield CPA. It has an
absorption maximum at 430 nm and smaller absorption
bands at 530 and 565 nm and presents an excitation spectra
similar to that of the fluorophore above described. This fact
probably indicates that other indolocarbazoles are being
produced during the cultivation.
Acknowledgments
The authors acknowledge Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) for
financial support, Dorival Martins for his support with
C. violaceum and Prof Dr Carol H. Collins for English
revision.
The compound represented by the dashed red line in
Figures 7b shows a broad excitation band at 400-470 nm and
peak emission around 512 nm. The concentration profile
indicates that this fluorophore is produced during violacein
biosynthesis. Finally, in Figure 7b, the excitation profile
represented by the dotted red line with narrow maximum
centered at 575 nm and an emission maximum at 675 nm
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The use of the multi-wavelength fluorescence
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FAPESP has sponsored the publication of this article.