Leveraging a Y. lipolytica naringenin chassis for biosynthesis of apigenin and associated glucoside.

Flavonoids are a diverse set of natural products with promising bioactivities including anti-inflammatory, anti-cancer, and neuroprotective properties. Previously, the oleaginous host Yarrowia lipolytica has been engineered to produce high titers of the base flavonoid naringenin. Here, we leverage this host along with a set of E. coli bioconversion strains to produce the flavone apigenin and its glycosylated derivative isovitexin, two potential nutraceutical and pharmaceutical candidates. Through downstream strain selection, co-culture optimization, media composition, and mutant isolation, we were able to produce168 mg/L of apigenin, representing a 46% conversion rate of 2-(R/S)-naringenin to apigenin. This apigenin platform was modularly extended to produce isovitexin by addition of a second bioconversion strain. Together, these results demonstrate the promise of microbial production and modular bioconversion to access diversified flavonoids.

Temporal sorting of microdroplets can identify productivity differences of itaconic acid from libraries of Yarrowia lipolytica.

Microdroplet screening of microorganisms can improve the rate of strain selection and characterization within the canonical design-build-test paradigm. However, a full analysis of the microdroplet environment and how well these conditions translate to culturing conditions and techniques is lacking in the field. Quantification of three different biosensor/analyte combinations at 12 hour timepoints reveals the potential for extended dose-response ranges as compared to traditional in vitro conditions. Using these dynamics, we present an application and analysis of microfluidic droplet screening utilizing whole-cell biosensors, ultimately identifying an altered productivity profile of itaconic acid in a Yarrowia lipolytica-based piggyBac transposon library. Specifically, we demonstrate that the timepoint for microdroplet selection can influence the outcome of the selection and thus shift the identified strain productivity and final titer. In this case, strains selected at earlier timepoints showed increased early productivity in flask scale, with the converse true as well. Differences in response indicate microdroplet assays require tailored development to more accurately sort for phenotypes that are scalable to larger incubation volumes. Likewise, these results further highlight that screening conditions are critical parameters for success in high-throughput applications.

Valorization of Caribbean Sargassum biomass as a source of alginate and sugars for de novo biodiesel production.

Since 2011, a massive influx of pelagic brown algae Sargassum has invaded coastlines causing environmental and economic disaster. Valorizing this plentiful macroalgae can present much needed economic relief to the areas affected. Here the production of biodiesel and a high-value alginate stream using Sargassum biomass collected from the coast of Quintana Roo, Mexico is reported. Biomass was pretreated via AEA (Alginate Extraction Autohydrolysis) and enzymatic saccharification via fungal Solid State Fermentation, releasing 7 g/L total sugars. The sugar mixture was fermented using engineered Yarrowia lipolytica resulting in 0.35 g/L total lipid titer at the lab tube scale. Additionally, the capability of extracting 0.3875 g/g DW of a high-value, purified alginate stream from this material is demonstrated. The findings presented here are promising and suggest an opportunity for the optimization and scale up of a biodiesel production biorefinery for utilization of Sargassum seaweeds during seasons of high invasion.

Progress in the metabolic engineering of bio-based lactams and their ω-amino acids precursors.

Lactams, cyclic carboxamide acids, are important building blocks as monomers for the manufacture of polyamides (nylons), with a market of millions of tons per year. Likewise, their non-natural building blocks, straight chain ω-amino acids, also have a wide range of applications as pharmaceuticals, therapeutic agents, and precursors to other platform chemicals. Current industrial lactam production requires petrochemically-derived routes that involve the use of harsh chemicals and reaction conditions. Microbial production provides a more sustainable method for production, from cost effective renewable resources. This review provides an extensive overview of progress toward the microbial production of lactams, particularly 4C butyrolactam, 5C valerolactam and 6C caprolactam, and their ω-amino acid precursors. Additionally, recent advances in the field as well as proposed microbial production pathways will be discussed, as well as future perspectives for the production of these important bulk chemicals.