From lab to market: An integrated bioprocess design approach for new-to-nature biosurfactants produced by Starmerella bombicola.

Glycolipid microbial biosurfactants, such as sophorolipids (SLs), generate high industrial interest as 100% biobased alternatives for traditional surfactants. A well-known success story is the efficient SL producer Starmerella bombicola, which reaches titers well above 200 g/L. Recent engineering attempts have enabled the production of completely new types of molecules by S. bombicola, e.g. the bolaform SLs. Scale-up of bolaform SL production was performed at 150 L scale. The purified product was evaluated in detergent applications, as classic SLs are mostly applied in eco-friendly detergents. In this paper, we show that they can be used as green and non-irritant surfactants in for example (automatic) dishwashing applications. However, due to the presence of an ester function in the biosurfactant molecule a limited chemical stability at higher pH values (>6.5) was noticed, (therefore called 'non-symmetrical' (nsBola)) which, is a major drawback that will most likely inhibit market introduction. An integrated bioprocess design (IBPD) strategy was thus applied to resolve this issue. The strategy was to replace the fed fatty acids with fatty alcohols, to generate so-called "symmetrical bolaform (sBola) sophorosides (SSs)," containing two instead of one glycosidic bond. Next to a change in feeding strategy, the blocking of the fatty alcohols from metabolizing/oxidizing through the suggested ω-oxidation pathway was necessary. For the latter, two putative fatty alcohol oxidase genes (fao1 and fao2) were identified in the S. bombicola genome and deleted in the bolaform SL producing strain (ΔatΔsble). Shake flask experiments for these new strains (ΔatΔsbleΔfao1 and ΔatΔsbleΔfao2) were performed to evaluate if the fed fatty alcohols were directly implemented into the SL biosynthesis pathway. Indeed, sBola sophorosides (SSs) production up to 20 g/L was observed for the ΔatΔsbleΔfao1 strain. Unexpectedly, the ΔatΔsbleΔfao2 strain only produced minor amounts of sBola sophorosides (SSs), and mainly nsBola SLs (alike the parental ΔatΔsble strain). The sBola sophorosides (SSs) were purified and their symmetrical structure was confirmed by NMR. They were found to be significantly more stable at higher pH, opening up the application potential of the biosurfactant by enhancing its stability properties.

Towards the industrialization of new biosurfactants: Biotechnological opportunities for the lactone esterase gene from Starmerella bombicola.

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.

Glucosomes: Glycosylated Vesicle-in-Vesicle Aggregates in Water from pH-Responsive Microbial Glycolipid.

Vesicle-in-vesicle self-assembled containers, or vesosomes, are promising alternatives to liposomes because of their possible hierarchical encapsulation and high stability. We report herein the first example of sugar-based vesicles-in-vesicles, which we baptize glucosomes. These were prepared by using a natural microbial glycolipid (branched C22 sophorolipid) extracted from the culture medium of the yeast Pseudohyphozyma bogoriensis. Glucosomes spontaneously formed in water between pH 6 and pH 4 at room temperature, without the requirement of any additive. By means of pH-resolved in situ small angle X-ray scattering, we provided direct evidence for the vesicle-formation mechanism. Statistical treatment of the vesicle radii distribution measured by cryo-tansmission electron microscopy by using a derived form of the Helfrich bending free-energy expression provided an order of magnitude for the effective bending constant (the sum of the curvature and the saddle-splay moduli) of the lipid membrane to K=(0.4±0.1) kBT. This value is in agreement with the bending constant measured for hydrocarbon-based vesicles membranes.