While rhamnolipids of the Pseudomonas aeruginosa type are commercially available, the natural diversity of rhamnolipids and their origin have barely been investigated. Here, we collected known and identified new rhlA genes encoding the acyltransferase responsible for the synthesis of the lipophilic rhamnolipid precursor 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA). Generally, all homologs were found in Betaproteobacteria and Gammaproteobacteria. A likely horizontal gene transfer event into Actinobacteria is the only identified exception. The phylogeny of the RhlA homologs from Pseudomonas and Burkholderia species is consistent with the organism phylogeny, and genes involved in rhamnolipid synthesis are located in operons. In contrast, RhlA homologs from the Enterobacterales do not follow the organisms’ phylogeny but form their own branch. Furthermore, in many Enterobacterales and Halomonas from the Oceanospirillales, an isolated rhlA homolog can be found in the genome. The RhlAs from Pseudomonas aeruginosa PA01, Pseudomonas fluorescens LMG 05825, Pantoea ananatis LMG 20103, Burkholderia plantarii PG1, Burkholderia ambifaria LMG 19182, Halomonas sp. strain R57-5, Dickeya dadantii Ech586, and Serratia plymuthica PRI-2C were expressed in Escherichia coli and tested for HAA production. Indeed, except for the Serratia RhlA, HAAs were produced with the engineered strains. A detailed analysis of the produced HAA congeners by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) highlights the congener specificity of the RhlA proteins. The congener length varies from 4 to 18 carbon atoms, with the main congeners consisting of different combinations of saturated or monounsaturated C10, C12, and C14 fatty acids. The results are discussed in the context of the phylogeny of this unusual enzymatic activity. IMPORTANCE The RhlA specificity explains the observed differences in 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) congeners. Whole-cell catalysts can now be designed for the synthesis of different congener mixtures of HAAs and rhamnolipids, thereby contributing to the envisaged synthesis of designer HAAs.

A novel whole cell cascade for double oxidation of cyclooctane to cyclooctanone was developed. The one-pot oxidation cascade requires only a minimum of reaction components: resting E. coli cells in aqueous buffered medium (=catalyst), the target substrate and oxygen as environmental friendly oxidant. Conversion of cyclooctane was catalysed with high efficiency (50% yield) and excellent selectivity (>94%) to cyclooctanone. The reported oxidation cascade represents a novel whole cell system for double oxidation of non-activated alkanes including an integrated cofactor regeneration. Notably, two alcohol dehydrogenases from Lactobacillus brevis and from Rhodococcus erythropolis with opposite cofactor selectivities and one monooxygenase P450 BM3 were produced in a coexpression system in one single host. The system represents the most efficient route with a TTN of up to 24363 being a promising process in terms of sustainability as well.

The structure−property relationship of rhamnolipids, RLs, well-known microbial bioamphiphiles (biosurfactants), is explored in detail by coupling cryogenic transmission electron microscopy (cryo-TEM) and both ex situ and in situ small-angle X-ray scattering (SAXS). The self-assembly of three RLs with reasoned variation of their molecular structure (RhaC10, RhaC10C10, and RhaRhaC10C10) and a rhamnose-free C10C10 fatty acid is studied in water as a function of pH. It is found that RhaC10 and RhaRhaC10C10 form micelles in a broad pH range and RhaC10C10 undergoes a micelle-to-vesicle transition from basic to acid pH occurring at pH 6.5. Modeling coupled to fitting SAXS data allows a good estimation of the hydrophobic core radius (or length), the hydrophilic shell thickness, the aggregation number, and the surface area per RL. The essentially micellar morphology found for RhaC10 and RhaRhaC10C10 and the micelle-to-vesicle transition found for RhaC10C10 are reasonably well explained by employing the packing parameter (PP) model, provided a good estimation of the surface area per RL. On the contrary, the PP model fails to explain the lamellar phase found for the protonated RhaRhaC10C10 at acidic pH. The lamellar phase can only be explained by values of the surface area per RL being counterintuitively small for a di-rhamnose group and folding of the C10C10 chain. These structural features are only possible for a change in the conformation of the di-rhamnose group between the alkaline and acidic pH.

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