Increasing Uniformity of Biosurfactant Production in Starmerella bombicola via the Expression of Chimeric Cytochrome P450s

Abstract

Sophorolipids are one of the best known microbial biosurfactants and are produced by several yeast species. The best studied producer is Starmerella bombicola, a non-pathogenic yeast associated in nature with bumblebees. Sophorolipids are built up of the rare disaccharide sophorose, which is attached to a fatty acid through a glyosidic bound. Sophorolipids produced by S. bombicola mainly contain oleic acid as the incorporated hydrophobic group. Other chain lengths can, to a certain content, be incorporated by feeding the yeast with substrates of alternative chain lengths. However, the efficiency for such substrates is low as compared to the preferred C18 chain length and defined by the substrate specificity of the first enzymatic step in sophorolipid biosynthesis, i.e., the cytochrome P450 enzyme CYP52M1. To increase product uniformity and diversity at the same time, a new strain of S. bombicola was developed that produces sophorolipids with a palmitic acid acyl chain. This was achieved by heterologous expression of the cytochrome P450 cyp1 gene of Ustilago maydis and feeding with palmitic acid. Optimization of the production was done by protein and process engineering.

Palmitic acid sophorolipid biosurfactant: from self-assembled fibrillar network (SAFiN) to hydrogels with fast recovery

“…The molecule was hydrolysed under alkaline conditions and only the non-acetylated acidic form of SLC16:0 was collected and used in this study. The detailed conditions of biosynthesis, purification as well as the full HPLC-ELSD, LC-MS and NMR characterization are provided in [52]. The typical 1 H NMR spectrum of the compound used in this work is reported in the electronic supplementary material, figure S1.…”

Microbial Synthesis of Nonionic Long‐Chain Sophorolipid Emulsifiers Obtained from Fatty Alcohol and Mixed Lipid Feeding

“…NMR spectra were recorded with a 400 MHz Bruker Ascend 400 in deuterated dimethyl sulfoxide (DMSO-d6). Besides 1 H-und 13 C-NMR analysis, 2D-NMR experiments were used for structural analysis. Homonuclear bond correlation was elucidated with COSY experiments, heteronuclear bond correlation with HSQC, and HMBC experiments.…”

“…While these are hydroxylated by a P450 monooxygenase and directly incorporated into the SLs, substrates of other chain lengths are either elongated or metabolized via the -oxidation pathway. [11][12][13] Only Rhodotorula bogoriensis is known to synthesize a C22 SL, which possesses a branched chain lipid tail hydroxylated at position C13. [14,15] So far only a few attempts focused on the production of more hydrophobic straight-chain SLs, which may be useful as bio-based emulsifiers.…”

“…A variety of fatty alcohols was tested and additionally equimolar fatty acid/fatty alcohol mixtures were examined as substrates. We hypothesized that the P450 monooxygenase [13] catalyzed hydroxylation of the fatty acid is preferred over fatty alcohol oxidation. Therefore production of SLs with tailored lipids by feeding oleic acid in combination with fatty alcohols directly to the microorganisms ( Figure 1E,F) should be a feasible alternative to chemical SL modification.…”

“…The preferred substrates for the yeasts are C16 and C18 fatty acids. While these are hydroxylated by a P450 monooxygenase and directly incorporated into the SLs, substrates of other chain lengths are either elongated or metabolized via the β‐oxidation pathway . Only Rhodotorula bogoriensis is known to synthesize a C22 SL, which possesses a branched chain lipid tail hydroxylated at position C13 …”