Biology subjects

Barriuso, J.

Publications and source records attributed to Barriuso, J..

8 recordsLinked to original sources

bioRxiv2026

Integrated valorization of glycerol and PET into lipids and PHAs using an engineered Yarrowia lipolytica strain and a Pseudomonas-Comamonas consortium

The accumulation of plastic waste necessitates innovative strategies that convert polymer carbon into value-added products. Here, we present a sequential yeast-bacterial workflow for the integrated valorisation of glycerol and amorphous polyethylene terephthalate (amPET) into triacyl glyceride (TAGs) and polyhydroxyalkanoates (PHAs). First, an engineered obese strain of Yarrowia lipolytica was cultivated on glycerol, for the simultaneous production of intracellular lipids, up to 42.9% of its cell dry weight, and secretion of a PET-depolymerizing enzymatic cocktail, composed of the cutinase from Mycothermus thermophilus (HiC) and the lipase B from Moesziomyces antarcticus (CALB). The resulting enzymatic crude hydrolysed amPET, and the released degradation products--terephthalic acid (TPA) and ethylene glycol (EG)-- served as feedstocks for a bacterial consortium composed of Comamonas testosteroni RW31 and Pseudomonas putida JM37, which naturally assimilate TPA and EG, respectively. This consortium successfully upcycled the released monomers into intracellular polyhydroxybutyrate (PHB) and medium-chain-length PHAs. Furthermore, fluorescent strains of both bacteria enabled the development of a semi-quantitative method for monitoring the consortium population dynamics. Overall, this study provides a robust proof-of-concept for a circular bioeconomy approach, successfully coupling glycerol-based enzyme and lipid production with the downstream biological conversion of PET-derived monomers into valuable bioplastics. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/729029v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1abe237org.highwire.dtl.DTLVardef@190a675org.highwire.dtl.DTLVardef@1a06dfaorg.highwire.dtl.DTLVardef@1945fe6_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2026

Integrated valorisation of PET and xylose using the oleaginous microorganisms Yarrowia lipolytica and Rhodococcus jostii

The increasing accumulation of polyethylene terephthalate (PET) waste has prompted the development of sustainable biotechnological strategies for its degradation and valorisation. This study presents an integrated approach combining enzymatic PET depolymerization by Yarrowia lipolytica, engineered to express and secrete the cutinase HiC and the lipase CalB, with the microbial valorization of PET-derived monomers, terephthalic acid (TPA) and ethylene glycol (EG), by Rhodococcus jostii RHA1. Y. lipolytica was further engineered for xylose metabolism, enabling enzyme production from low-cost lignocellulose-derived substrates. Enzymatic assays with HiC and CalB crudes effectively hydrolysate PET to TPA and EG, demonstrating functional enzymatic activity without purification steps. In addition, R. jostii RHA1 was able to use as substrate the released monomers and accumulated intracellular lipids. Overall, this work demonstrates the feasibility of coupling the production of PET degrading enzymes and microbial lipid, using an abundant monosaccharide, with the assimilation of the PET degradation products to also produce microbial lipids. This modular system provides a promising framework for the sustainable upcycling of plastic waste into value-added bioproducts within a circular economy.

synthetic biology↗

bioRxiv2026

AI-assisted improvement of Aspergillus oryzae β-galactosidase using an Ensemble of Protein Language Models

{beta}-galactosidases (BGs) are essential enzymes widely used in the food industry, particularly in the production of lactose-free products. Among them, the BG from Aspergillus oryzae is of industrial relevance due to its activity at acidic pH and moderate thermal tolerance. However, enhancing its catalytic performance remains a key challenge. Traditional enzyme engineering methods are time-consuming and resource-intensive, limiting their scalability. Recent advances in Artificial Intelligence (AI), particularly those based on Natural Language Processing, offer a promising alternative by enabling efficient exploration of protein sequence space and prediction of beneficial mutations. In this study, we introduce an ensemble-based, zero-shot Protein Language Model pipeline that reconciles predictions from six independent models (ESM2 and the five ESM1v variants) combined with a diversity-aware candidate selection strategy. Applied to the BG from A. oryzae, this approach identified beneficial mutations leading to novel enzyme variants with up to a four-fold increase in catalytic efficiency on oNPGal, a two-fold increase on lactose, and, independently, a T338I variant with markedly enhanced thermostability ({approx}80% residual activity after 24 h at 60 {degrees}C), all without requiring supervised fine-tuning on experimental fitness data. Our results demonstrate that consensus across an ensemble of PLMs can efficiently enrich beneficial substitutions in industrially relevant enzymes and substantially reduce the number of wet-lab candidates that need to be screened. Table of Contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/726739v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@16477baorg.highwire.dtl.DTLVardef@f071c5org.highwire.dtl.DTLVardef@1bd7777org.highwire.dtl.DTLVardef@1ee2999_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2026

Integrating Fungal-Bacterial Synergy to Enhance Circular MFC-Hydroponic Performance

Microbial fuel cells (MFCs) represent a promising technology for the simultaneous treatment of wastewater and bioelectricity generation. In this study, the MFCs are conceived as functional modules to be integrated into hydroponic cultivation systems, acting as a prosthetic rhizosphere capable of coupling wastewater treatment and bioelectrochemical activity with plant nutrition improvement. We compared the electrochemical performance of different microbial consortia comprising the electroactive bacterium Shewanella oneidensis, the plant growth promoting rhizobacterium (PGPR) Pseudomonas putida, and the plant biomass-degrading fungus Ophiostoma piceae, along with the supplementation with the quorum sensing (QS) analogue molecule 1{square} dodecanol. These microbial consortia are tested in MFCs fed with wastewater and root exudates to analyze enhanced feedstock assimilation, electricity production, and the generation of plant growth-promoting substances (PGPS). From an electrochemical perspective, we evaluated planktonic growth, anode adhesion, substrate consumption, and the production of redox-active molecules and PGPS such as flavins and siderophores respectively alongside key electrical production parameters, including current output and power. Among the different microbial configurations tested, the consortium combining S. oneidensis, P. putida, and O. piceae exhibited the highest electrical production potential. Moreover, within this framework, we detected the extracellular production of siderophores in MFCs containing P. putida, suggesting a potential role supporting hydroponic crop growth. Furthermore, the addition of 1-dodecanol led to an improvement of the bioelectrochemical parameters. These results highlight the potential of synthetic microbial consortia in MFC-based systems not only to enhance electricity generation from wastewater but also to provide added value in integrated hydroponic applications through rhizosphere-like functions.

microbiology↗

bioRxiv2025

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

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.

synthetic biology↗

bioRxiv2025

Biochemical Upcycling of PET via Glycolysis and Engineered Microbial Consortia

Polyethylene terephthalate (PET) waste remains a major environmental challenge due to its recalcitrance and low economic value. Here, we present an integrated biochemical approach that couples glycolysis with a synthetic microbial consortium to upcycle post-consumer PET (pcPET) into polyhydroxyalkanoates (PHA). Glycolysis efficiently depolymerized pcPET into bis(2-hydroxyethyl) terephthalate (BHET) in 2 h, circumventing the limitations of in vivo PET degradation. We engineered a two-species microbial consortium composed of Comamonas testosteroni RW31, able to metabolise terephthalic acid, and Pseudomonas putida JM37, able to consume ethylene glycol, each modified for the extracellular secretion of PET- and MHET-hydrolases, employing different plasmid architectures. This division of labour enabled rapid BHET hydrolysis and the subsequent upcycling of the released monomers into PHAs. The combination of the different strains allowed to select C. testosteroni pSEVA354-MHETase and P. putida pSEVA234-PETase as the best consortium, based on growth and PHAs content. Overall, this work proposes a strategy for PET waste depolymerisation and valorisation, highlighting the potential of mixed chemical and biological approaches and the use of non-conventional microbial chassis within engineered consortia.

synthetic biology↗

bioRxiv2025

Contrasting evolutionary forces of specialization and admixture underlie the genomic and phenotypic diversity of Yarrowia lipolytica

Microbial diversity emerges from evolutionary processes that shape genomic and phenotypic traits in response to complex environmental pressures. Deciphering these dynamics is key to understanding microbial ecology and advancing biotechnological applications. Here, we use the yeast Yarrowia lipolytica to study genomic signatures of adaptation across a broad range of environments and to illustrate how population-level data can inform targeted bioprospecting for industrial traits. Whole-genome and phenotypic analyses of 126 isolates from natural and anthropogenic environments reveal a complex population structure in this species, shaped by both niche specialization and admixture events. Structured lineages exhibit ecological filtering, reduced genetic diversity, and distinct gene content, consistent with adaptation to substrates like dairy, hydrocarbons, or industrial substrates. In contrast, admixed populations display greater genetic diversity and broader phenotypic capacity, including enhanced stress tolerance and metabolic flexibility. Genome plasticity, reflected in pangenome and CNV variation, aligns with ecological origin, while trait assays link phenotypic divergence to underlying genetic variation. For instance, better growth performance on acetate, an ecological and industrially relevant trait, is associated with hydrocarbon-adapted strains and likely linked to variation in regulatory regions of acetate metabolism genes. Together, our results reflect how divergent evolutionary trajectories--ranging from ecological specialization to genomic plasticity through admixture-- underpin the species ecological success and provide a framework for harnessing its natural diversity in microbial bioprospecting.

evolutionary biology↗

bioRxiv2025

Pseudomonas putida JM37 as a novel bacterial chassis for ethylene glycol upcycling

Ethylene glycol (EG), one of the main monomers of polyethylene terephthalate (PET), is an attractive target for microbial upcycling. Despite this interest, there is a limited number of described organisms that can efficiently metabolise EG. Here, we report the metabolic and biotechnological potential of Pseudomonas putida JM37 as a novel bacterial chassis for EG valorization. We show that JM37 efficiently grows on EG as the sole carbon and energy source, outperforming other Pseudomonas strains. Genome sequencing and directed mutagenesis revealed that genetic redundancies in the glyoxylate assimilation pathways underlie its robust EG metabolism. Beyond biomass generation, we demonstrated the biotechnological potential of JM37. This strain was able to accumulate medium-chain polyhydroxyalkanoates (mcl-PHAs), dominated by C10 monomers, directly from EG. Moreover, JM37 successfully expressed heterologous biosynthetic pathways, including a violacein biosynthetic operon and a PET-hydrolase which has been secreted actively into the extracellular medium. Together, our results support the use of P. putida JM37 as a versatile synthetic biology chassis for sustainable EG upcycling and as a promising platform for circular bioproduction.

synthetic biology↗