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bioRxiv · 10.64898/2025.12.19.695487

Synthetic Yarrowia lipolytica consortium for efficient conversion of lignocellulosic oligosaccharides into lipids

Crespo-Roche, D.·Gonzalez-Garcia, D.·Mendez-Liter, J. A.·Ledesma-Amaro, R.·Prieto, A.·Barriuso, J.
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Abstract

Lignocellulosic biomass (LCB) is an abundant and renewable feedstock for the sustainable production of bioproducts; however, its industrial exploitation is limited by its complex composition and by the lack of microbial platforms capable of simultaneously degrading and assimilating cellulose- and hemicellulose-derived oligosaccharides. Yarrowia lipolytica lacks the native enzymatic machinery required for this process. In this study, we engineered a multifunctional strain (YBXT-XR-BGL3) able to secrete fungal {beta}-glucosidase (BGL3 or BGL1) and {beta}-xylosidase (BxTw1) from Talaromyces amestolkiae, enabling the hydrolysis of cellobiose and xylooligosaccharides, respectively. In addition, a xylose reductase pathway was introduced to confer xylose assimilation. Because the construction of a single multifunctional strain may impose a significant metabolic burden and reduce fitness, we benchmarked this strain against a division-of-labor strategy. To this end, we also developed a cellobiose-specialized strain (YBGL3 or YBGL1) and a xylooligosaccharide-specialized strain (YBXT-XR). Functional characterization revealed efficient saccharification of cello- and xylooligosaccharides under acidic conditions, with BGL3 outperforming BGL1 in glucose release and BxTw1 exhibiting broad pH tolerance. Under nitrogen-limited conditions, this enabled lipid accumulation of up to 20% from cellobiose in YBGL3 and YBXT-XR-BGL3, and up to 15% from xylooligosaccharides in YBXT and YBXT-XR-BGL3. In co-culture experiments using a mixed substrate (glucose, cellobiose, and xylooligosaccharides), both the multifunctional strain and the consortium produced up to 0.67 g L{square}{superscript 1} of lipids. However, the division-of-labor approach led to higher lipid accumulation (34% versus 26.3% in the monoculture), driven by a rapid population shift: following cellobiose depletion (after 72 h), YBXT-XR became predominant and utilized the remaining xylooligosaccharides almost exclusively for lipid synthesis. Overall, this study provides the first demonstration of a Y. lipolytica system capable of simultaneously utilizing cellulose- and hemicellulose-derived oligosaccharides. Moreover, benchmarking a division-of-labor consortium against a multifunctional monoculture highlights a robust strategy to enhance lipid biosynthesis and improve process resilience for LCB valorization.

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Crespo-Roche, D., Gonzalez-Garcia, D., Mendez-Liter, J. A., Ledesma-Amaro, R., Prieto, A., Barriuso, J.. 2025-12-22. Synthetic Yarrowia lipolytica consortium for efficient conversion of lignocellulosic oligosaccharides into lipids. https://doi.org/10.64898/2025.12.19.695487

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