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.
Glycosylation of small molecules can significantly alter their properties such as solubility, stability, and/or bioactivity, making glycosides attractive and highly demanded compounds. Consequently, many biotechnological glycosylation approaches have been developed, with enzymatic synthesis and whole-cell biocatalysis as the most prominent techniques. However, most processes still suffer from low yields, production rates and inefficient UDP-sugar formation. To this end, a novel metabolic engineering strategy is presented for the in vivo glucosylation of small molecules in Escherichia coli W. This strategy focuses on the introduction of an alternative sucrose metabolism using sucrose phosphorylase for the direct and efficient generation of glucose 1-phosphate as precursor for UDP-glucose formation and fructose, which serves as a carbon source for growth. By targeted gene deletions, a split metabolism is created whereby glucose 1-phosphate is rerouted from the glycolysis to product formation (i.e., glucosylation). Further, the production pathway was enhanced by increasing and preserving the intracellular UDP-glucose pool. Expression of a versatile glucosyltransferase from Vitis vinifera (VvGT2) enabled the strain to efficiently produce 14 glucose esters of various hydroxycinnamates and hydroxybenzoates with conversion yields up to 100%. To our knowledge, this fast growing (and simultaneously producing) E. coli mutant is the first versatile host described for the glucosylation of phenolic acids in a fermentative way using only sucrose as a cheap and sustainable carbon source.
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