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

Abstract

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.

Unraveling and resolving inefficient glucolipid biosurfactants production through quantitative multiomics analyses of Starmerella bombicola strains

“…The strain produced 118 g/L GLs (compared to 8.38 g/L for the ∆ ugtB1 :: URA3 G9 strain, data not shown). The productivity over the entire process amounted up to 0.51 g·L −1 ·h −1 for the ∆ ugtB1 :: URA3 PT36 strain (compared to 0.01 g·L −1 ·h −1 for the ∆ ugtB1 :: URA3 G9 strain, data not shown), which is in line with the general glycolipid productivity values (between 0.5 and 1 g·L −1 ·h −1 ) of other engineered S. bombicola strains (Roelants et al, ; Van Bogaert et al, ; Van Renterghem et al, ). With the original strain we were not able to produce high amounts of GLs, so the application testing remained very limited as mentioned in the introduction section (Baccile, Cuvier, et al, ; Baccile, Selmane et al, ).…”

“…The final titer of the original ∆ ugtB1 :: URA3 G9 strain, amounted to only 3 g/L, while that of the ∆ ugtB1 :: URA3 PT36 strain reached 27 g/L (Figure a). This was similar to glycolipid production values for other engineered S. bombicola strains as for the production of acidic SL's (Roelants et al, ), nonsymmetrical bolaform sophorolipids (Van Bogaert et al, ) and symmetrical bolaform sophorosides (Van Renterghem et al, ).…”

“…Of these GLs (mix non‐ and mono‐acetylated GLs) a portion was deacetylated similarly as described to generate nonacetylated acidic SLs (Roelants et al, ) and purified products were thus generated as depicted in Figure . It must be stressed that this is a very straightforward, green, and scalable DSP method compared to the methods that were described for other glycolipid molecules (Van Renterghem et al, ). This will allow easier valorization of these molecules in the future.…”

“…The cultures were sampled at regular intervals and analyzed for several parameters; optical density, glucose concentration, and colony‐forming units as described before (Roelants et al, ; Saerens, Saey, et al, ). Sophorolipid and glucolipid analyses were performed by “ultra high‐performance liquid chromatography—evaporative light scattering detector” (UPLC‐ELSD) as described by Van Renterghem et al ().…”

“…This yeast also has inherent potential as “chassis” organism for the production of other industrially relevant (glyco)lipid compounds, due to its highly efficient native glycolipid machinery, efficient acetyl‐CoA, and uridine diphosphate glucose (UDP)‐glucose machinery and its resistance to (high amounts) of hydrophobic/antibiotic compounds (Van Bogaert, De Maeseneire, Develter, Soetaert, & Vandamme, ; Van Bogaert, Fleurackers, Van Kerrebroeck, Develter, & Soetaert, ) and efficient uptake/secretion systems for hydrophobic and amphiphilic compounds. In the past, the natural SL production of this yeast production has already been extended toward the production of different (new‐to‐nature) biochemicals by strain engineering (Roelants et al, ; Roelants et al, ; Soetaert, De Maeseneire, Saerens, Roelants, & Van Bogaert, ; Van Bogaert, Buyst, Martins, Roelants, & Soetaert, ; Van Renterghem et al, ). However, some of these novel strains are showing severe productivity drops compared with the very efficient wild type SL production (Roelants et al, ; Saerens, Saey, & Soetaert, ; Saerens, Zhang, Saey, Van Bogaert, & Soetaert, ) without clear cause.…”