Edible algal biomass has emerged as a promising platform for nutritional bioproduct development, but its practical translation depends on compositional variability, intake feasibility, and safety constraints. Here, we integrated biochemical profiling, trace element screening, age-specific intake modeling, and community consumption context to evaluate commercially available and supplier-provided microalgal and seaweed products within realistic application boundaries. Selected products differed substantially in macronutrient composition, fatty acid profiles, pigments, polyphenols, and mineral composition, indicating product- and species-specific roles as nutritional biomass rather than interchangeable ingredients. Across species and age groups, approximately 0.7 to 3.1 g dry biomass provided 20

The auxin-inducible degron (AID) system enables targeted protein degradation in vivo. Conventionally, the AID tag is fused directly to the terminus of the target protein; however, its intrinsically disordered nature and destabilizing motifs can render it susceptible to premature proteolytic removal from its tagged protein, resulting in incomplete degradation upon auxin induction. In addition, direct terminal fusion of the conventional AID tag may compromise protein stability. To address these limitations, we introduce a new degron design in which the conventional AID tag, mIAA7, is inserted into an exposed loop of green fluorescent protein (GFP). The resulting engineered GFP variant, termed the "constrained AID" (cAID) tag, was validated in the industrially important oleaginous yeast Yarrowia lipolytica. Inserting mIAA7 into GFP, rather than fusing it directly at the GFP terminus, elevated expression of the GFP fusion protein and enabled more complete degradation upon auxin induction. The utility of the novel cAID tag was further validated by tagging a soluble cytosolic anti-mCherry nanobody and by targeting β-carotene ketolase, squalene synthase, and squalene epoxidase to modulate carotenoid biosynthesis. Compared to direct fusion with the conventional AID tag, cAID tag enabled more complete protein degradation and tighter temporal control of metabolism, while minimizing perturbation to the tagged protein.

The production of multiple proteins using a single open reading frame (sORF)/polyprotein system is a powerful strategy for coordinated multi-protein expression in eukaryotes. The most widely used approach relies on 2A peptides, but conventional 2A systems suffer from several limitations, and their viral origin makes them less than ideal for commercial crop biotechnology applications. Self-excising protein (SEP) modules are a promising alternative that enables coordinated production of multiple proteins from a single ORF encoding a polyprotein precursor. SEPs provide distinct advantages over conventional systems and effectively address many of the limitations inherent to the 2A approach. An SEP module is a fusion protein composed of an N-terminal excising domain (NED) and a C-terminal excising domain (CED) joined by a peptide linker. Using this architecture, a panel of SEP modules has been developed by pairing an engineered intein (serving as the NED) with various CEDs, including a second engineered intein, a 2A-like peptide, and ubiquitin. These modules release multiple proteins from the polyprotein precursor with nearly stoichiometric expression and clean cleavage. Coordinated coexpression using SEPs has been successfully demonstrated in several eukaryotic systems, including yeast, mammalian cells, and plants. This review offers a comprehensive analysis of the SEP technology while underscoring its major applications.

Biohybrid robots and biohybrid robot-on-a-chip have been developed for drug screening and toxicity screening the complementing animal experiments. A sensing system is needed to evaluate the response to external stimulation, but until now, only the human motor system-based biohybrid robot-on-a-chip with neuromuscular system has been developed without a sensing system. A human nervous system-based biohybrid robot-on-a-chip with eye function as a sensing system in addition to brain/motor neuron/muscle functions is proposed for the first time. Eye assembloid is fabricated by a combination of thalamic organoid covered with Au nanomesh and four retinal organoids. By a combination of eye assembloid, cerebral organoid, motor neuron spheroid, and muscle bundle on polymer substrate, a human nervous system-based biohybrid robot-on-a-chip is made. When blue light is used to cause retinal damage or a hydroxychloroquine (HCQ), a retinal toxic chemical, is applied to the eye assembloid, they caused a decrease in muscle bundle contraction. These results indicated that electrophysiological signals generated by the eye assembloid are transmitted to the muscle bundle through cerebral organoid and motor neuron spheroids, and thus the proposed system can perform the toxicity screening. Human nervous system-based biohybrid robot-on-a-chip can be applied to drug screening of neurodegenerative diseases and toxicity screening for the complement of animal experiments in the future.

Yarrowia lipolytica is an industrial yeast that can convert waste oil to value-added products. However, it is unclear how this yeast metabolizes lipid feedstocks, specifically triacylglycerol (TAG) substrates. This study used 13C-metabolic flux analysis (13C-MFA), genome-scale modeling, and transcriptomics analyses to investigate Y. lipolytica W29 growth with oleic acid, glycerol, and glucose. Transcriptomics data were used to guide 13C-MFA model construction and to validate the 13C-MFA results. The 13C-MFA data were then used to constrain a genome-scale model (GSM), which predicted Y. lipolytica fluxes, cofactor balance, and theoretical yields of terpene products. The three data sources provided new insights into cellular regulation during catabolism of glycerol and fatty acid components of TAG substrates, and how their consumption routes differ from glucose catabolism. We found that (1) over 80% of acetyl-CoA from oleic acid is processed through the glyoxylate shunt, a pathway that generates less CO2 compared to the TCA cycle, (2) the carnitine shuttle is a key regulator of the cytosolic acetyl-CoA pool in oleic acid and glycerol cultures, (3) the oxidative pentose phosphate pathway and mannitol cycle are key routes for NADPH generation, (4) the mannitol cycle and alternative oxidase activity help balance excess NADH generated from β-oxidation of oleic acid, and (5) asymmetrical gene expressions and GSM simulations of enzyme usage suggest an increased metabolic burden for oleic acid catabolism.

Bio-solar cells are studied as sustainable and biocompatible energy sources with significant potential for biomedical applications. However, they are composed of light-harvesting biomolecules with narrow absorption wavelengths and weak transient photocurrent generation. In this study, a nano-biohybrid-based bio-solar cell composed of bacteriorhodopsin, chlorophyllin, and Ni/TiO2 nanoparticles is developed to overcome the current limitations and verify the possibility of biomedical applications. Bacteriorhodopsin and chlorophyllin are introduced as light-harvesting biomolecules to broaden the absorption wavelength. As a photocatalyst, Ni/TiO2 nanoparticles are introduced to generate a photocurrent and amplify the photocurrent generated by the biomolecules. The developed bio-solar cell absorbs a broad range of visible wavelengths and generates an amplified stationary photocurrent density (152.6 nA cm(-2)) with a long lifetime (up to 1 month). Besides, the electrophysiological signals of muscle cells at neuromuscular junctions are precisely regulated by motor neurons excited by the photocurrent of the bio-solar cell, indicating that the bio-solar cell can control living cells by signal transmission through other types of living cells. The proposed nano-biohybrid-based bio-solar cell can be used as a sustainable and biocompatible energy source for the development of wearable and implantable biodevices and bioelectronic medicines for humans.

Conditional protein degradation is a powerful tool for controlled protein knockdown. The auxin-inducible degron (AID) technology uses a plant auxin to induce depletion of degron-tagged proteins, and it has been shown to be functional in several non-plant eukaryotes. In this study, we demonstrated AID-based protein knockdown in an industrially important oleaginous yeast Yarrowia lipolytica. Using the mini-IAA7 (mIAA7) degron derived from Arabidopsis IAA7, coupled with an Oryza sativa TIR1 (OsTIR1) plant auxin receptor F-box protein (expressed from the copper-inducible MT2 promoter), C-terminal degron-tagged superfolder GFP could be degraded in Yarrowia lipolytica upon addition of copper and the synthetic auxin 1-Naphthaleneacetic acid (NAA). However, leaky degradation of the degron-tagged GFP in the absence of NAA was also noted. This NAA-independent degradation was largely eliminated by replacing the wild-type OsTIR1 and NAA with the OsTIR1F74A variant and the auxin derivative 5-Ad-IAA, respectively. Degradation of the degron-tagged GFP was rapid and efficient. However, Western blot analysis revealed cellular proteolytic cleavage within the mIAA7 degron sequence, leading to the production of a GFP sub-population lacking an intact degron. The utility of the mIAA7/OsTIR1F74A system was further explored in controlled degradation of a metabolic enzyme, β-carotene ketolase, which converts β-carotene to canthaxanthin via echinenone. This enzyme was tagged with the mIAA7 degron and expressed in a β-carotene producing Y. lipolytica strain that also expressed OsTIR1F74A controlled by the MT2 promoter. By adding copper and 5-Ad-IAA at the time of culture inoculation, canthaxanthin production was found to be reduced by about 50% on day five compared to the control culture without adding 5-Ad-IAA. This is the first report that demonstrates the efficacy of the AID system in Y. lipolytica. Further improvement of AID-based protein knockdown in Y. lipolytica may be achieved by preventing proteolytic removal of the mIAA7 degron tag.

Simultaneous coexpression of multiple proteins is essential for biotechnology and synthetic biology. Currently, the most popular polyprotein coexpression system utilizes the foot-and-mouth disease virus (FMDV) 2A peptide that mediates translational ribosome-skipping events. However, due to unfavorable consumer acceptance of transgenic products containing animal-virus sequences, novel non-viral 2A-like peptides from purple sea urchin (Strongylcentrotus purpuratus) and California sea slug (Aplysia californica) were investigated for polyprotein coexpression in this study. We demonstrated that these non-viral 2A sequences functioned similarly to their viral counterpart in polyprotein processing, in both plant and mammalian cells, and were successfully used to express a functional recombinant antibody. The new non-viral 2A-like sequences offer an alternative tool for engineering multigenic traits or production of protein complexes as biomedicine via coexpression of protein subunits.

This study aimed to develop a bioprocess using plant oil as the carbon source for lipid-assimilating yeast to produce high-value astaxanthin. Using high-oleic safflower oil as a model, efficient cell growth and astaxanthin production by the engineered Yarrowia lipolytica strain ST7403 was demonstrated, and a considerable portion of astaxanthin was found excreted into the spent oil. Astaxanthin was the predominant carotenoid in the extracellular oil phase that allowed facile in situ recovery of astaxanthin without cell lysis. Autoclaving the safflower oil medium elevated the peroxide level but it declined quickly during fermentation (reduced by 84% by day 3) and did not inhibit cell growth or astaxanthin production. In a 1.5-L fed-batch bioreactor culture with a YnB-based medium containing 20% safflower oil, and with the feeding of casamino acids, astaxanthin production reached 54 mg/L (53% excreted) in 28 days. Further improvement in astaxanthin titer and productivity was achieved by restoring leucine biosynthesis in the host, and running fed-batch fermentation using a high carbon-to-nitrogen ratio yeast extract/peptone medium containing 70% safflower oil, with feeding of additional yeast extract/peptone, to attain 167 mg/L astaxanthin (48% excreted) in 9.5 days of culture. These findings facilitate industrial microbial biorefinery development that utilizes renewable lipids as feedstocks to not only produce high-value products but also effectively extract and recover the products, including non-native ones. Key Points • Yarrowia lipolytica can use plant oil as a C-source for astaxanthin production. • Astaxanthin is excreted and accumulated in the extracellular oil phase. • Astaxanthin is the predominant carotenoid in the extracellular oil phase. • Plant oil serves as a biocompatible solvent for in situ astaxanthin extraction.

Yarrowia lipolytica has emerged as an important non-model host for terpene production. However, three main challenges remain in industrial production using this yeast. First, considerable knowledge gaps exist in metabolic flux across multiple compartments, cofactor generation, and catabolism of non-sugar carbon sources. Second, many enzymatic steps in the complex-terpene synthesis pathway can pose rate-limitations, causing accumulation of toxic intermediates and increased metabolic burdens. Third, metabolic shifts, morphological changes, and genetic mutations are poorly characterized under industrial fermentation conditions. To overcome these challenges, systems metabolic analysis, protein engineering, novel pathway engineering, model-guided strain design, and fermentation optimization have been attempted with some successes. Further developments that address these challenges are needed to advance the Yarrowia lipolytica platform for industrial-scale production of high-value terpenes, including those with highly complex structures such as anticancer molecules withanolides and insecticidal limonoids.

This study employs biomass growth analyses and 13C-isotope tracing to investigate lipid feedstock utilization by Yarrowia lipolytica. Compared to glucose, oil-feedstock in the minimal medium increases the yeast's biomass yields and cell sizes, but decreases its protein content (<20% of total biomass) and enzyme abundances for product synthesis. Labeling results indicate a segregated metabolic network (the glycolysis vs. the TCA cycle) during co-catabolism of sugars (glucose or glycerol) with fatty acid substrates, which facilitates resource allocations for biosynthesis without catabolite repressions. This study has also examined the performance of a β-carotene producing strain in different growth mediums. Canola oil-containing yeast-peptone (YP) has resulted in the best β-carotene titer (121 ± 13 mg/L), two-fold higher than the glucose based YP medium. These results highlight the potential of Y. lipolytica for the valorization of waste-derived lipid feedstock.

Efficient coproduction of multiple proteins or their subunits is important in many facets of life sciences and biotechnology. Here, we report a novel approach that exploits the synergy between an engineered mini-intein and an ubiquitin variant to achieve coordinated coexpression of multiple proteins in eukaryotic hosts, from a single open reading frame that encodes a polyprotein precursor consisting of proteins of interest (POIs) connected by an intervening intein-ubiquitin fusion domain. The intein variant mediates highly active autocatalytic cleavage at its N-terminus, whereas the endogenous deubiquitinases cleave at ubiquitin's C-terminus, leading to the release of the POIs. Using fluorescent reporter proteins for proof-of-concept, utility of the intein-ubiquitin domain was validated in higher plants and yeast systems. Essentially complete release of the POIs was achieved as demonstrated with western blots. Proteins expressed using the intein-ubiquitin system potentially preserve their intended sequences, which is important for preventing alteration of POI function.

Papaya (Carica papaya) is widely cultivated in many tropical regions of the world. With an estimated 30-50% cull rate, there is a large amount of off-grade papaya produced. Here, we report very low-cost processing of culled papaya fruit waste, without needing any complex mechanized operations, to yield several products, including seed oil, sugar-rich puree, detoxified/defatted seed meal, and crude myrosinase and glucosinolates with antimicrobial and biofumigation applications. We then demonstrated that both puree and seed oil can serve as effective carbon substrates for cultivation of the oleaginous yeast Yarrowia lipolytica to produce single-cell proteins and high-value recombinant protein products. To use papaya seed oil for culturing Y. lipolytica, the concentration of the inhibitory substance benzyl isothiocyanate (BITC) in the oil needs to be minimized. If the culled fruits (and hence seeds) were stored frozen prior to drying, a very high level (>30 mM) of BITC was detected in the oil extracted from the dried seeds. However, if the seeds were not frozen prior to drying, oil from dried papaya seeds contained almost no BITC, and could support vigorous growth of Y. lipolytica, with efficient production of a functional nanobody fusion protein at a level similar to that achieved using olive oil. By using both juice and seed lipid, rather than juice alone, Y. lipolytica biomass produced per unit papaya more than doubled. As Y. lipolytica is amenable to genetic manipulation, and is known as a proficient cell factory with many industrial applications, the papaya waste valorization technology could potentially be extended to produce additional useful products such as biofuel and oleochemicals from Y. lipolytica.

In the present study, we fabricated magnetic oleosomes functionalized with recombinant proteins as a new carrier for oil-based lipophilic drugs for cancer treatment. The bioengineered oleosome is composed of neutral lipids surrounded by a phospholipid monolayer with embedded oleosin fusion proteins. The oleosin was genetically fused to a nanobody of a green fluorescent protein (GFP). A recombinant protein consisting of immunoglobulin-binding protein LG fused to GFP was used to couple the oleosome to an antibody for targeted delivery to breast cancer cells. The lipid core of the oleosome was loaded with magnetic nanoparticles and carmustine as the lipophilic drug. The magnetic oleosome was characterized using transmission electron microscopy and dynamic light scattering. Moreover, the specific delivery of oleosome into the target cancer cell was investigated via confocal microscopy. To examine the cell viability of the delivered oleosome, a conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was carried out. Furthermore, an animal study was conducted to confirm the effect resulting from the delivery of the anticancer drug-loaded oleosomes. Taken together, the fabricated lipophilic drug-loaded magnetic oleosome can be a powerful tool for oil-based drug delivery agent for cancer therapy.

In this study, extended artificial scaffoldins possessing multiple cohesin modules were created in vivo by employing split-intein-mediated protein ligation. Artificial scaffoldins having one Clostridium thermocellum cohesin (Coht), one carbohydrate binding module (CBM) from Clostridium cellulolyticum scaffolding protein CipC, and one to five cohesins (Cohc) derived from CipC, were assembled. These scaffoldins were used to assemble cellulosomal enzyme complexes for investigating the interplay among endoglucanase, exoglucanase, and scaffoldin-borne CBM, on the hydrolysis of a model microcrystalline cellulose substrate, Avicel. The cellulosomal complexes were assembled in vitro by incubating recombinant C. thermocellum endoglucanase (At) and C. cellulolyticum exoglucanase (Ec), with the various artificial scaffoldins. Under a fixed total cellulase concentration, improved hydrolysis is noted by recruiting both Ec and At on the same scaffoldin, for all scaffoldins tested, compared with free cellulases. The improvement is more profound with scaffoldins having a higher Cohc/Coht ratio (i.e., increased Ec/At ratio). Furthermore, among scaffoldins having the same Cohc/Coht ratio, highest rates of Avicel hydrolysis are noted when Coht, and hence an endoglucanase, is situated next to the CBM and not flanked by Cohc. These results point to the importance of using scaffoldins with sufficiently high numbers of cohesin units to achieve an optimal exo-/endo-glucanase ratio to create efficient designer cellulosomes. Furthermore, intein-trans-splicing is proven here to be an effective method for assembling complex scaffoldins and more intricate cellulosomes.

A novel approach is developed for coordinated expression of multiple proteins from a single transgene in plants. An Ssp DnaE mini-intein variant engineered for hyper-N-terminal autocleavage is covalently linked to the foot-and-mouth disease virus 2A (F2A) peptide with unique ribosome skipping property, via a peptide linker, to create an 'IntF2A' self-excising fusion protein domain. This IntF2A domain acts, in cis, to direct highly effective release of its flanking proteins of interest (POIs) from a 'polyprotein' precursor in plants. This is successfully demonstrated in stably transformed cultured tobacco cells as well as in different organs of transgenic tobacco plants. Highly efficient polyprotein processing mediated by the IntF2A domain was also demonstrated in lettuce and Nicotiana benthamiana based on transient expression. Protein constituents released from the polyprotein precursor displayed proper function and accumulated at similar levels inside the cells. Importantly, no C-terminal F2A extension remains on the released POIs. We demonstrated co-expression of as many as three proteins in plants without compromising expression levels when compared with those using single-protein vectors. Accurate differential cellular targeting of released POIs is also achieved. In addition, we succeeded in expressing a fully assembled and functional chimeric anti-His Tag antibody in N. benthamiana leaves. The IntF2A-based polyprotein transgene system overcomes key impediments of existing strategies for multiprotein co-expression in plants, which is particularly important for gene/trait stacking.

Low cost electric power generation using raw juice from culled papaya or fruit waste as feedstock was achieved in a sugar-air alkaline battery (SAAB) with about 37% coulombic efficiency at 0.7–0.8 mW cm−2 near peak power density. Such fruit waste, juice-based electrochemical energy could be poised as sustainable distributed energy resource for remote or underdeveloped regions that need cheap electricity without complicated infrastructure.

Alzheimer's disease (AD) is the most common brain disorder worldwide. Aberrant tau hyperphosphorylation and accumulation play critical roles in the formation of neurofibrillary tangles highly associated with neuronal dysfunction and cognitive impairment in AD pathogenesis. Glycogen synthase kinase-3β (GSK3β) is a key kinase responsible for tau hyperphosphorylation. Selective inhibition of GSK3β is a promising strategy in AD therapy. Corn silks (CS, Zea mays L.) have been traditionally used as a medicinal herb and recently noted for their potentially cognitive benefits. However, the neuroprotective components of CS and their molecular mechanism have received little attention to date. As part of our effort screening phytochemicals against a broad panel of kinases targeting AD tauopathy, we found inhibition of GSK3β by CS extracts. Subsequent bioassay-guided fractionation led to the isolation and identification of two 6-C-glycosylflavones, isoorientin (1) and 3'-methoxymaysin (2), with selective inhibition against GSK3β in vitro. Enzyme kinetics and molecular docking studies demonstrated that 1 specifically inhibited GSK3β via an ATP noncompetitive mechanism, acting as a substrate competitive inhibitor of GSK3β. Further in vitro cellular studies demonstrated that 1 effectively attenuated tau phosphorylation mediated by GSK3β and was neuroprotective against β-amyloid-induced tau hyperphosphorylation and neurotoxicity in SH-SY5Y cells. The C-glycosylflavones represent new lead candidates with a novel mechanism of action for the development of AD phytopharmaceuticals.

The present study reports CO2 capture and conversion to bicarbonate using Escherichia coli expressing carbonic anhydrase (CA) on its cell surface in a novel foam bioreactor. The very large gas-liquid interfacial area in the foam bioreactor promoted rapid CO2 absorption while the CO2 in the aqueous phase was subsequently converted to bicarbonate ions by the CA. CO2 gas removal in air was investigated at various conditions such as gas velocity, cell density and CO2 inlet concentration. Regimes for kinetic and mass transfer limitations were defined. Very high removal rates of CO2 were observed: 9570gCO(2)m(bioreactor)(-3)h(-1) and a CO2 removal efficiency of 93% at 4% inlet CO2 when the gas retention time was 24s, and cell concentration was 4g(dw)L(-1). These performances are superior to earlier reports of experimental bioreactors using CA for CO2 capture. Overall, this bioreactor system has significant potential as an alternative CO2 capture technology.

Being able to coordinate co-expression of multiple proteins is necessary for a variety of important applications such as assembly of protein complexes, trait stacking, and metabolic engineering. Currently only few options are available for multiple recombinant protein co-expression, and most of them are not applicable to both prokaryotic and eukaryotic hosts. Here, we report a new polyprotein vector system that is based on a pair of self-excising mini-inteins fused in tandem, termed the dual-intein (DI) domain, to achieve synchronized co-expression of multiple proteins. The DI domain comprises an Ssp DnaE mini-intein N159A mutant and an Ssp DnaB mini-intein C1A mutant connected in tandem by a peptide linker to mediate efficient release of the flanking proteins via autocatalytic cleavage. Essentially complete release of constituent proteins, GFP and RFP (mCherry), from a polyprotein precursor, in bacterial, mammalian, and plant hosts was demonstrated. In addition, successful co-expression of GFP with chloramphenicol acetyltransferase, and thioredoxin with RFP, respectively, further substantiates the general applicability of the DI polyprotein system. Collectively, our results demonstrate the DI-based polyprotein technology as a highly valuable addition to the molecular toolbox for multi-protein co-expression which finds vast applications in biotechnology, biosciences, and biomedicine.