Ignacio Moya-Ramírez

Synthetic microbial consortia have a significant potential for novel biotechnological applications such as the upgrade of complex feedstocks. However, achieving stable and reproducible co-cultures is challenging due to competitive dynamics and unbalanced growth rates among species. Here, we present an effective method for microbial encapsulation relying on alginate core-shell hydrogel beads coated with ε-poly-L-lysine (εPLL-HB). This procedure ensures complete containment of several microbial species (prokaryotic and eukaryotic) while allowing for consistent growth inside the beads. Compared to chitosan and α-poly-L-lysine, two of the biomaterials most used as coating agents for this kind of encapsulation, εPLL demonstrated superior performance in avoiding cellular escape across diverse culture conditions and for all the microbial strains tested. εPLL-HB enabled the construction of spatially distributed co-cultures, effectively balancing populations between microorganisms with different growth rates. Furthermore, microbial encapsulation inside εPLL-HB provided protection against toxic compounds in lignocellulosic-derived media and maintained the encapsulation efficacy and cell viability after long-term storage at −80 °C. The superior microbial containment, structural integrity and chemical resistance of εPLL-HB, combined with their cost-effective and simple preparation, makes them a versatile tool for synthetic microbial consortia engineering, with broad applicability in biotechnological processes.

Spent coffee grounds (SCG) are wastes generated in high amounts worldwide. Their composition makes them a promising feedstock for biotechnological processes. Here we show that the production of the biosurfactant surfactin by submerged culture of a Bacillus subtilis strain growing on SCG is possible, reaching concentrations up to 8.8 mg/L when using SCG at 8.3 g/L in the medium. In addition, we report a synergy between the production of surfactin and the recovery of melanoidins, an added-value compound already present in SCG. More specifically, the concentration of melanoidins in the culture medium increased between 2.1 and 2.5 times thanks to the presence of the B. subtilis in the culture. Furthermore, we have observed a strong interaction between surfactin and melanoidin aggregates through dynamic light scattering measurements, and that both of them can be co-purified with an acid precipitation. We have also characterized the interfacial and antioxidant properties of the cell-free supernatant and surfactin extract, as well as the distribution of the congeners of the biosurfactant. Altogether, this work describes a promising approach to obtain biosurfactants and antioxidant molecules in a single operation, which can be used to design several new formulations of interest for bioremediation, amendment of soils, food and cosmetics.

Glycosylation is a critical post-translational modification of proteins, improving properties such as folding, half-life and functionality. However, glycosylation is a non-templated and heterogeneous process because of the promiscuity of the enzymes involved. Here we describe a platform for s eq u ential g lycosyl a tion r eactions for ta ilo r ed su g ar s t ructures (SUGAR-TARGET) that allows bespoke, controlled N-linked glycosylation in vitro . This novel proof-of-concept system is enabled by immobilised enzymes produced with a “one-step immobilisation/purification” method to express, biotinylate in vivo and immobilise glycosyltransferases. The immobilised enzymes are used in a reaction cascade mimicking a human-like N-linked glycosylation pathway where promiscuity naturally exists. The enzyme cascade is applied to free glycans, and a monomeric Fc domain expressed in glycoengineered Pichia pastoris , yielding near homogeneous glycoforms (>95% conversion). Finally, immobilised β-1,4 galactosyltransferase is used to enhance the galactosylation profile of three different IgGs yielding 80.2 – 96.3 % terminal galactosylation. Enzyme recycling was further demonstrated for 7 cycles, with a combined reaction time greater than 140 hours. The novel SUGAR-TARGET platform is easy to implement, modular and reusable, and therefore can lead to the development of homogeneous glycan structures for functional and clinical evaluation. The use of immobilised enzymes enables the economical modification of cell-based material supporting applications at a large industrial scale.

Coexistence of different populations of cells and isolation of tasks can provide enhanced robustness and adaptability or impart new functionalities to a culture. However, generating stable cocultures involving cells with vastly different growth rates can be challenging. To address this, we developed living analytics in a multilayer polymer shell (LAMPS), an encapsulation method that facilitates the coculture of mammalian and bacterial cells. We leverage LAMPS to preprogram a separation of tasks within the coculture: growth and therapeutic protein production by the mammalian cells and l-lactate biosensing by Escherichia coli encapsulated within LAMPS. LAMPS enable the formation of a synthetic bacterial–mammalian cell interaction that enables a living biosensor to be integrated into a biomanufacturing process. Our work serves as a proof-of-concept for further applications in bioprocessing since LAMPS combine the simplicity and flexibility of a bacterial biosensor with a viable method to prevent runaway growth that would disturb mammalian cell physiology.

Probing the energy landscape and thermodynamics of biomolecules for drug design.

BACKGROUND: Waste oils are gaining interest as an alternative carbon source in fermentation processes as an option for the development of cost‐effective and renewable processes. In this study, the production of the biosurfactant surfactin by three different Bacillus subtilis strains was investigated using waste frying oils as carbon source in two different forms: (i) emulsified waste oil (EWO), and (ii) emulsified and enzymatically hydrolyzed waste oil (HWO). Thus, the objective of this work is to analyze the effect of a hydrolytic pretreatment on the increase in the bioavailability of the waste oil over the cell metabolism and surfactin yields.RESULTS: Ultra‐performance liquid chromatography mass spectrometry (UPLC‐MS) analyses confirmed that the biosurfactant produced by all three strains is surfactin, the congener distribution being dependent on the strain and medium composition. Although the emulsification of the waste oil led to an increase in biomass production, surfactin concentration did not improve significantly, whatever the strain used. However, HWO shifted the cell metabolism towards the production of the surfactin congener C15 (with 15 carbon atoms on its hydrophobic chain) almost exclusively.CONCLUSIONS: The reported behavior of B. subtilis strains growing on hydrolyzed frying oil could be of interest for the production of pure surfactin congeners from waste materials. © 2019 Society of Chemical Industry

Waste frying oils (WFO) can be both environmental pollutants and a source of valuable products. In this work we explore the conversion of WFO into surface‐active substances, such as FFA and partial glycerides, through enzymatic hydrolysis in O/W emulsion. Two different WFO and three lipases of different origin were tested. In addition, we optimized the conditions for the production of O/W emulsions to be used as reaction medium as well as several reaction parameters, such as the type of enzyme and its concentration, the pH, and the presence of Ca²⁺ ions. Gum arabic‐stabilized emulsions with an oil fraction of 0.15 proved to be the most adequate owing to their high interfacial area and short‐term stability. The physicochemical characterization of both WFO revealed the presence of an increased amount of surface‐active matter relative to food‐grade vegetable oils. These substances, mainly FFA, can interfere with lipase action and reduce the reaction rate. However, the extent of hydrolysis was only slightly affected by them, and remained fairly similar to that achieved with a control mixture of food‐grade vegetable oils. The product distribution depended on the enzyme used. Pseudomonas fluorescens lipase was found to be particularly suitable for the production of partial glycerides (mono‐ and diacylglycerols), which are in great demand by the food industry. In any case our results demonstrate that WFO are a good substrate for enzymatic hydrolysis, comparable to food‐grade vegetable oils, providing an alternative route for the valorization of this waste.