Bubbling insights: unveiling the true sophorolipid biosynthetic pathway by Starmerella bombicola.

BACKGROUND: The yeast Starmerella bombicola is renowned for its highly efficient sophorolipid production, reaching titers and productivities of (over) 200 g/L and 2 g/(L h), respectively. This inherent efficiency has led to the commercialization of sophorolipids. While the sophorolipid biosynthetic pathway has been elucidated a few years ago, in this study, it is revisited and true key intermediates are revealed. RESULTS: Recently, Starmerella bombicola strains developed and evaluated in the past were reevaluated unveiling unexpected findings. The AT enzyme encoded in the sophorolipid biosynthetic gene cluster is the only described enzyme known to acetylate sophorolipids, while the SBLE enzyme encoded by the SBLE gene is described to catalyze the conversion of (acetylated) acidic sophorolipids into lactonic sophorolipids. A double knockout of both genes was described to result in the generation of bolaform sophorolipids. However, new experiments performed with respective S. bombicola strains Δsble, Δat Δsble, and ∆at revealed inconsistencies with the current understanding of the SL pathway. It was observed that the ∆sble strain produces mainly bolaform sophorolipids with higher acetylation degrees instead of acidic sophorolipids. Furthermore, the ∆at strain produces predominantly bolaform sophorolipids and lactonic sophorolipids with lower acetylation degrees, while the ∆at ∆sble strain predominantly produces bolaform sophorolipids with lower acetylation degrees. These results indicate that the AT enzyme is not the only enzyme responsible for acetylation of sophorolipids, while the SBLE enzyme performs an intramolecular transesterification reaction on bolaform glycolipids instead of an esterification reaction on acidic sophorolipids. These findings, together with recent in vitro data, led us to revise the sophorolipid biosynthetic pathway. CONCLUSIONS: Bolaform sophorolipids instead of acidic sophorolipids are the key intermediates in the biosynthetic pathway towards lactonic sophorolipids. Bolaform sophorolipids are found in very small amounts in extracellular S. bombicola wild type broths as they are very efficiently converted into lactonic sophorolipids, while acidic sophorolipids build up as they cannot be converted. Furthermore, acetylation of sophorolipids is not exclusively performed by the AT enzyme encoded in the sophorolipid biosynthetic gene cluster and acetylation of bolaform sophorolipids promotes their transesterification. These findings led to the revision of the industrially relevant sophorolipid biosynthetic pathway.

Revision of the sophorolipid biosynthetic pathway in Starmerella bombicola based on new insights in the substrate profile of its lactone esterase.

BACKGROUND: Sophorolipids (SLs) are a class of natural, biodegradable surfactants that found their way as ingredients for environment friendly cleaning products, cosmetics and nanotechnological applications. Large-scale production relies on fermentations using the yeast Starmerella bombicola that naturally produces high titers of SLs from renewable resources. The resulting product is typically an extracellular mixture of acidic and lactonic congeners. Previously, we identified an esterase, termed Starmerella bombicola lactone esterase (SBLE), believed to act as an extracellular reverse lactonase to directly use acidic SLs as substrate. RESULTS: We here show based on newly available pure substrates, HPLC and mass spectrometric analysis, that the actual substrates of SBLE are in fact bola SLs, revealing that SBLE actually catalyzes an intramolecular transesterification reaction. Bola SLs contain a second sophorose attached to the fatty acyl group that acts as a leaving group during lactonization. CONCLUSIONS: The biosynthetic function by which the Starmerella bombicola 'lactone esterase' converts acidic SLs into lactonic SLs should be revised to a 'transesterase' where bola SL are the true intermediate. This insights paves the way for alternative engineering strategies to develop designer surfactants.

Kinetic resolution of sec-alcohols catalysed by Candida antarctica lipase B displaying Pichia pastoris whole-cell biocatalyst.

Kinetic resolution of sec-alcohols is a green process with biocatalyst. Candida antarctica lipase B (CALB) displayed on Pichia pastoris cell-surface (Pp-CALB) was characterized in kinetic resolution of sec-alcohols with different structures. The reaction parameters including acyl donors, molar ratio of substrates, solvents and temperatures were examined with 2-octanol as model substrate. 47.4% molar conversion of 2-octanol and 99.7% eep were obtained after a 5h reaction with Pp-CALB, and 90% of its original activity still remained after being reused for 10 cycles. Pp-CALB was then used to several sec-alcohols and it showed great enzymatic activity and enantioselectivity to all tested sec-alcohols, more than 93.1% of eep. The enantioselective characteristics of Pp-CALB catalysed sec-alcohols with different structures were compared with Novozyme 435 which was almost the same. Solvent free system as one way of green chemistry was applied to Pp-CALB and Pp-CALB showed great catalytic activity and enantioselectivity. Pp-CALB was potential biocatalyst of high enzymatic activity and enantioselectivity using in resolution of sec-alcohols.

Enzymatic Process Enhances the Flavour Profile and Increases the Proportion of Esters in Citrus Essential Oils.

Citrus essential oils (CEOs) are important flavors in the food and confectionary industries. A lipase process was proposed for enhancing the flavor profiles and increasing the proportions of esters in CEOs. The effects of the enzymatic process were explored by detecting the constituents of the CEOs of American sweet orange oil (ASO) and Brazil mandarin oil (BMO) through GC/MS and sensory evaluation by a trained panel, and positive effects were confirmed by both methods. A further eleven kinds of CEOs were treated via the lipase process and increments of 10 - 1170% were achieved in the proportions of esters, which were mostly ethyl esters. Enhancement in fruity odor, especially the top note, was demonstrated by all CEOs after enzymatic processing. All CEOs were tested for antimicrobial activities, and only ASO displayed fairly ideal antimicrobial activities. Meanwhile, modified ASO showed a certain increase in antimicrobial activities. This methodology might be considered a sustainable route for acquiring 'natural' essential oils with enhanced flavor profiles and simultaneously enhancing the comprehensive utilization of citrus fruits.