High rhamnolipids production correlates with a high intracellular R- to Sspecific enoyl-CoA hydratase activity Ahmad M. Abdel-Mawgoud* and Eric Déziel email@example.com 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.