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High rhamnolipids production correlates with a high intracellular R- to Sspecific enoyl-CoA hydratase activity
Ahmad M. Abdel-Mawgoud* and Eric Déziel
ahmad.saleh@iaf.inrs.ca
INRS - Institut Armand-Frappier, Laval, Québec, Canada, H7V 1B7
Abstract
Outline
AcylCoA
Rhamnolipids (RLs) are multipurpose surface-active molecules produced from rhamnose and
R-3-hydroxyalkanoate (C10±2) precursors by the pathogenic bacterium Pseudomonas
aeruginosa.
Interestingly, 3-hydroxyalkanoate precursors of RLs are exclusively in the R-form and we
recently reported that they originate from enoyl-CoA (C10±2) intermediates of β-oxidation
diverted by the action of R-specific enoyl-CoA hydratase (R-ECH), called RhlY/Z.
S- and R-ECH catalyze the stereospecific hydration of 2-enoyl-CoA into the corresponding Sand R-3-hydroxyacyl-CoA, respectively.
In this study, we investigated the correlation between the net R-/S-ECH activity of P.
aeruginosa PA14 in comparison with different bacteria under various culture conditions using
our recently developed chiral HPLC method coupled with tandem mass spectrometry (MS) for
the stereospecific and quantitative analysis of S-/R-3-hydroxyalkanoates.
We show that, in mineral salts medium designed for high RLs production, the net ECH activity
of P. aeruginosa is almost exclusively of the R-specific type (R-ECH). On the other hand, in
rich medium conditions characterized by low RLs production, a nearly equal S- to R-ECH
activities was observed.
We also evaluated the net ECH activity in two other bacteria that are candidates for the
heterologous expression of rhamnolipids genes, namely, Escherichia coli and Pseudomonas
putida, and found that the former is of mainly S-ECH type whether in rich or minimal media,
while the latter, like P. aeruginosa, is of equal R-/S-ECH in rich medium and of R-ECH in
mineral medium.
These observations could explain the reported failures to express RLs production in E. coli
and its success in P. putida. This study reveals an important analytical tool for evaluation of
the candidacy of different hosts for the heterologous expression of RLs, preferred in nonpathogenic hosts for commercial purposes, based on the nature of the net R-/S-ECH
activities and consequently the chiral profile of R-/S-3-hydroxyalkanoate precursor pool.
R-ECH
β-oxidation
FadB
Trans-2-enoylCoA
e.g. RhlYZ1
FadB
3-ShydroxyacylCoA
(ECH/I)
enoyl-CoA
Hydratase/isomerase
High RL production is
associated with high Rspecific ECH activity
1
S-3-hydroxyacyl-CoA
(Not An RL precursor)
S-ECH
3-RhydroxyacylCoA
RhlA
HAA
RhlB
/dTDP-L-rhamnose
R-3-hydroxyacyl-CoA
(An RL precursor)
Enoyl-CoA Hydratase
Trans-2-enoyl-CoA
FadE
FadA
3-ketoacylCoA
In the minimal
medium condition,
designed for high
rhamnolipids
production,
Pseudomonas
aeruginsoa showed
high R-specific ECH
activity
Abdel-Mawgoud, A. M., Lépine, F. & Déziel, E. A
novel pathway diverts β-oxidation intermediates to
the biosynthesis of rhamnolipid biosurfactants.
Chem Biol Under review (2013).
Mono
-RL
Results-1
Methodology
HPLC/MS-MS method for estimation of R-/S-ECH activities 2
R-specific ECH activity is in synchrony with RL production level by P.
aeruginosa in different culture conditions
In vitro ECH reaction using clarified total cell lysate and derivatization of
ECH reaction products
A
B
3000
Rhamnolipids conc (mg/L)
Intracellular ECH activity in rich and
minimal media
Percentage of R-/S-enantiomers of 3hydroxydecanoate
Rhamnolipid production in rich and
minimal media by PA14 strain
2500
2000
1500
1000
500
R-3-OH-C10
110
S-3-OH-C10
100
90
80
70
60
50
40
30
20
10
0
0
PA14 in
TSB
PA14 in
TSB
PA14 in
MSM
PA14 in
MSM
Figure 1. Rhamnolipids production by P. aeruginosa PA14 in optimized minimal salts medium (MSM) is
about 20 times that in rich Tryptic Soy Broth (TSB) medium (Fig. 1A). Synchronously, the in vitro R-specific
ECH activity (supplying R-3-hydroxyacyl-CoA precursors of RL) in cells of P. aeruginosa PA14 cultivated in
MSM is two times higher than that when cultivated in rich medium (Fig. 1B)
Results-2
Chiral lipid profiling as a tool for identification of candidate heterologous hosts
for RL expression: e.g. P. putida KT2440
Racemic R-/S-3-hydroxydecanoic acid:
90
Relative abundance
SRGraphical
representation
Percentage of R-/S- 3hydroxydecanoate
Rt: 18.35 min
Area: 5835
100
Rt: 21.16 min
Area: 5918
80
70
R
60
R-3-OH-C10
50
S-3-OH-C10
40
S
10
R-3-OH-C10
B
S-3-OH-C10
80
100
80
60
40
20
70
60
50
40
30
20
10
0
BL21/
LB
DH5α/
LB
PA14/
TSB
P.putida/
TSB
P.
fluorescens/
TSB
P. putida-pUCP26empty
/TSB
P. putida-pUCP26rhlAB
/TSB
Abdel-Mawgoud, A. M., Lépine, F. & Déziel, E. A chiral high-performance liquid chromatography–tandem mass spectrometry method
for the stereospecific analysis of enoyl-coenzyme A hydratases/isomerases. Journal of Chromatography A 1306, 37-43 (2013).
Figure 2. In vitro assay of R-/S-ECH activities in total cell lysates of different strains shows variable degrees
of R-specific ECH activities which is null in E. coli BL21 and detectable in P. putida (KT2440) (Fig. 2A).
Accordingly, the latter strain is rather suggested to be supplying lipid precursors of RL and hence a probable
candidate host for RL expression as proved experimentally in Fig. 2B.
Acknowledgements
Conclusions
0
Time
2
120
0
30
20
Percentage of R-/S-enantiomers of 3hydroxydecanoate
A
Rhamnolipids concentration (mg/L)
Chiral chromatographic separation and
tandem MS detection of derivatized
ECH reaction product
Racemic
This study was supported by NSERC Discovery grant No. 312478 to E.D.
A.M.A-M. is recipient of a Vanier - Canada Graduate Scholarship, and of a
tuition fees grant from the Ministry of Higher Education and Research, Egypt.
E.D. holds a Canada Research Chair.
• We demonstrate an efficient tool of chiral HPLC-tandem MS to estimate the net ECH activity in cell
lysates and determine its type (R-/S-specificity).
• For maximum and/or heterologous expression of rhamnolipids, it is a prerequisite that the host is naturally
supplying the two precursors of Rhamnolipids (RL); L-rhamnose and R-3-hydroxyalkanoate.
• The provided tool helps in the identification of the physiological conditions with maximal supply of the
latter precursor, R-3-hydroxyalkanoate, for maximum production of RL.
• Finally, the method is useful in the identification of potential candidate hosts for heterologous expression
of RL based on the nature of their R-specific ECH activity.
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