Although sophorolipids (SLs) produced by S. bombicola are a real showcase for the industrialization of microbial biosurfactants, some important drawbacks are associated with this efficient biological process, e.g., the simultaneous production of acidic and lactonic SLs. Depending on the application, there is a requirement for the naturally produced mixture to be manipulated to give defined ratios of the components. Recently, the enzyme responsible for the lactonization of SLs was discovered. The discovery of the gene encoding this lactone esterase (sble) enabled the development of promising S. bombicola strains producing either solely lactonic (using a sble overexpression strain described in this paper: oe sble) or solely acidic SLs (using a sble deletion strain, which was recently described, but not characterized yet: Δsble). The new S. bombicola strains were used to investigate the production processes (fermentation and purification) of either lactonic or acidic SLs. The strains maintain the high inherent productivities of the wild-type or even perform slightly better and thus represent a realistic industrial opportunity. 100% acidic SLs with a mixed acetylation pattern were obtained for the Δsble strain, while the inherent capacity to selectively produce lactonic SLs was significantly increased (+42%) for the oe sble strain (99% lactonic SLs). Moreover, the regulatory effect of citrate on lactone SL formation for the wild-type was absent in this new strain, which indicates that it is more robust and better suited for the industrial production of lactonic SLs. Basic parameters were determined for the purified SLs, which confirm that the two new strains produce molecules with distinctive properties of which the application potential can now easily be investigated independently.
Sophorolipids are among the best-positioned microbial biosurfactants to reach large-scale industrial production and application. However, the structural variety of sophorolipids offered by wild-type strains is rather limited, requiring their efficient modification to expand the application areas of sophorolipids. A combination of genetic engineering and green chemical modification via ozonolysis was applied in this work to produce key precursors useful in the development of a library of sophorolipid derivatives. Uniform symmetrical α,ω-bola sophorosides, produced by a novel strain of Starmerella bombicola, were investigated as substrates for the first time to generate 100% ω-C9 sophorosides (key precursors in the development of a sophorolipid library) via ozonolysis in water. Ozonolysis yielded a mixture of C9:0 ω-sophoroside aldehydes and C9:0 ω-sophorolipid acids. The selectivity toward the C9:0 ω-sophoroside aldehyde was increased using catalase, limiting the overoxidation of the aldehyde by the in situ formed H2O2. The C9:0 ω-sophorolipid acid could be produced selectively by extending the ozonolysis time. Moreover, using water as the solvent during ozonolysis proved to be beneficial in suppressing the formation of ozonides, therefore eliminating the need to perform a reductive or oxidative workup. Consequently, an efficient, safe, and scalable route has been established for the production of key sophoroside precursors.
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.
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