Monorhamnolipids and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) production using Escherichia coli as a heterologous host

Abstract

Pseudomonas aeruginosa produces the biosurfactants rhamnolipids and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs). In this study, we report the production of one family of rhamnolipids, specifically the monorhamnolipids, and of HAAs in a recombinant Escherichia coli strain expressing P. aeruginosa rhlAB operon. We found that the availability in E. coli of dTDP-L: -rhamnose, a substrate of RhlB, restricts the production of monorhamnolipids in E. coli. We present evidence showing that HAAs and the fatty acid dimer moiety of rhamnolipids are the product of RhlA enzymatic activity. Furthermore, we found that in the recombinant E. coli, these compounds have the same chain length of the fatty acid dimer moiety as those produced by P. aeruginosa. These data suggest that it is RhlAB specificity, and not the hydroxyfatty acid relative abundance in the bacterium, that determines the profile of the fatty acid moiety of rhamnolipids and HAAs. The rhamnolipids level produced in recombinant E. coli expressing rhlAB is lower than the P. aeruginosa level and much higher than those reported by others in E. coli, showing that this metabolic engineering strategy lead to an increased rhamnolipids production in this heterologous host.

RhlA Converts β-Hydroxyacyl-Acyl Carrier Protein Intermediates in Fatty Acid Synthesis to the β-Hydroxydecanoyl-β-Hydroxydecanoate Component of Rhamnolipids inPseudomonas aeruginosa

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]acetate (specific activity, 56 mCi/mmol), ␤-hydroxy[1-…”

“…C]decanoyl-CoA (specific activity, 55 Ci/mol), and [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]octanoyl-CoA (specific activity, 50 mCi/mmol) were obtained from American Radiolabeled Chemicals, Inc., and [2][3][4][5][6][7][8][9][10][11][12][13][14] C]malonyl-CoA (specific activity, 52 mCi/mmol) was obtained from Amersham Pharmacia Biotech. ACP and the His-tagged fatty acid biosynthetic enzymes were purified by affinity chromatography followed by gel filtration chromatography as described previously (5,10).…”

“…P. aeruginosa and E. coli strains were grown in M9 medium at 30°C to stationary phase, and cell-free supernatants were collected by centrifugation and filtered through 0.22-m membranes. Radioactive HAA was prepared from E. coli strain BL21(DE3) with pKZ003, grown to 5 ϫ 10 8 cells/ml and labeled with [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]acetate for 2 h before the cells were harvested. The supernatant was acidified to pH 2.0 and extracted twice with chloroform-methanol (2/1, vol/vol), and the lower organic phase was evaporated to dryness and resuspended in methanol.…”

Development of an Antibody for Detection of Rhamnolipids Characterized as a Major Bacterial Virulence Factor

“…Escherichia coli produces RLs in a very small amount, which is greatly increased when the bacterium is recombined with the operon rhlAB from bacterium P. aeruginosa [9]. Yet one more example is the thermophilic bacterium Thermus thermophilus HB8, which also produces RLs (TthRLs) in large amounts using sunflower seed oil, sodium gluconate or glucose as carbon source [10,11].…”

The Definition, Preparation and Application of Rhamnolipids as Biosurfactants

“…They have a glycosyl head group, in this case a rhamnose moiety, and a 3-(hydroxyalkanoyloxy) alkanoic acid (HAA) fatty acid tail [8][9]. Specifically there are two main classes of rhamnolipids, mono-rhamnolipids and di-rhamnolipids; consisting of one or two rhamnose groups respectively [10].…”