High-value Molecules Research Articles

Application of Rapeseed Meal Protein Isolate as a Supplement to Texture-Modified Food for the Elderly.

Rapeseed meal (RSM), a by-product of rapeseed oil extraction, is currently used for low-value purposes. With a biorefinery approach, rapeseed proteins may be extracted and recovered for high-end uses to fully exploit their nutritional and functional properties. This study reports the application of RSM protein isolate, the main output of a biorefining process aimed at recovering high-value molecules from rapeseed meal, as a supplement to texture-modified (TM) food designed for elderly people with mastication and dysphagia problems. The compositional (macronutrients by Official Methods of Analyses, and mineral and trace element profiles using Inductively Coupled Plasma Optical Emission Spectrometry ICP-OES), nutritional and sensory evaluations of TM chicken breast, carrots and bread formulated without and with RSM protein supplementation (5% w/w) are hereby reported. The results show that the texture modification of food combined with rapeseed protein isolate supplementation has a positive impact on the nutritional and sensory profile of food, meeting the special requirements of seniors. TM chicken breast and bread supplemented with RSM protein isolate showed unaltered or even improved sensory properties and a higher nutrient density, with particular regard to proteins (+20-40%) and minerals (+10-16%). Supplemented TM carrots, in spite of the high nutrient density, showed a limited acceptability, due to poor sensory properties that could be overcome with an adjustment to the formulation. This study highlights the potentialities of RSM as a sustainable novel protein source in the food sector. The application of RSM protein proposed here is in line with the major current challenges of food systems such as the responsible management of natural resources, the valorization of agri-food by-products, and healthy nutrition with focus on elderly people.

Microbial Carotenoid Synthesis Optimization in Goat Cheese Whey Using the Robust Taguchi Method: A Sustainable Approach to Help Tackle Vitamin A Deficiency.

The work describes the carotenoid synthesis process by Rhodotorula glutinis P4M422 using an agro-industrial waste as the substrate, seeking a biorefinery platform approach for waste utilization to produce high-value molecules. A culture medium based on goat milk whey (GMW) was optimized via the Taguchi method (L9 array). Four factors (ethanol, carbon and nitrogen source, and pH) were evaluated at three levels. The carbon and nitrogen composition were the factors dominating the process performance. Optimized conditions were validated (Urea, 0.3% w/v; pH, 4.5; ethanol, 10% v/v; glucose, 6.0%), and the carotenoid production (4075 µg/L) was almost 200% higher than when using the un-optimized process (2058 µg/L). Provitamin A carotenoids torulene, β-carotene, and γ-carotene (different proportions) were produced under all conditions. The hydrolyzed goat milk whey showed promising expectations as a low-cost source for carotenoid production by Rhodotorula glutinis P4M422. The results are important for the innovative sustainable production of carotenoid-rich matrices for different purposes (nutrition, health promotion, color) and industries (foods, nutricosmetics, nutraceuticals, feeds), notably to help to combat vitamin A deficiency.

Omics-guided bacterial engineering of Escherichia coli ER2566 for recombinant protein expression.

The goal of bacterial engineering is to rewire metabolic pathways to generate high-value molecules for various applications. However, the production of recombinant proteins is constrained by the complexity of the connections between cellular physiology and recombinant protein synthesis. Here, we used a rational and highly efficient approach to improve bacterial engineering. Based on the complete genome and annotation information of the Escherichia coli ER2566 strain, we compared the transcriptomic profiles of the strain under leaky expression and low temperature-induced stress. Combining the gene ontology (GO) enrichment terms and differentially expressed genes (DEGs) with higher expression, we selected and knocked out 36 genes to determine the potential impact of these genes on protein production. Deletion of bluF, cydA, mngR, and udp led to a significant decrease in soluble recombinant protein production. Moreover, at low-temperature induction, 4 DEGs (gntK, flgH, flgK, flgL) were associated with enhanced expression of the recombinant protein. Knocking out several motility-related DEGs (ER2666-ΔflgH-ΔflgL-ΔflgK) simultaneously improved the protein yield by 1.5-fold at 24°C induction, and the recombinant strain had the potential to be applied in the expression studies of different exogenous proteins, aiming to improve the yields of soluble form to varying degrees in comparison to the ER2566 strain. Totally, this study focused on the anabolic and stress-responsive hub genes of the adaptation of E. coli to recombinant protein overexpression on the transcriptome level and constructs a series of engineering strains increasing the soluble protein yield of recombinant proteins which lays a solid foundation for the engineering of bacterial strains for recombinant technological advances. KEY POINTS: • Comparative transcriptome analysis shows host responses with altered induction stress. • Deletion of bluF, cydA, mngR, and udp genes was identified to significantly decrease the soluble recombinant protein productions. • Synchronal knockout of flagellar genes in E. coli can enhance recombinant protein yield up to ~ 1.5-fold at 24°C induction. • Non-model bacterial strains can be re-engineered for recombinant protein expression.

Lignocellulose-degrading fungi newly isolated from central Morocco are potent biocatalysts for olive pomace valorization.

The investigation of lignocellulolytic catalysts is an important feature to face the challenges of lignocellulosic biomass valorization. In central Morocco, fungi were isolated from decaying wood, soil, olive crushing by-products and their compost. One hundred fifty-five isolates were submitted to a selective screening, which served to distinguish 83% of lignocellulolytic isolates. Then, a collection of 56 fungi was subjected to morphological and molecular identification with the ITS5 and ITS4 primers. This approach showed that 45% of the fungal population belonged to the genus Penicillium, followed by Aspergillus 14%, and Fusarium 11%. Alternaria, Trichoderma, Paecilomyces, Cladosporium, Trichocladium, Circinella, and Doratomyces genera are founded with a minority occurrence. Finally, validation of the enzymatic profile was done for 20 isolates, by testing their enzymatic performance on a liquid medium in the presence of cellulose, lignin, and olive pomace. The maximum protein production of 788µgml-1 was reached by an Alternaria strain, which produced also 10.6IUml-1 of endoglucanase. Thus, a β-glucosidase activity of 5.1IUml-1 was obtained by a Penicillium strain isolated from decaying wood. Regarding ligninolytic activities, olive pomace was the most suitable substrate to detect these activities. Decaying wood strains presented the most remarkable results with 1.1IUml-1, 0.7IUml-1 et 0.3IUml-1 for laccase, LiP and MnP, respectively. The use of the selected fungi and olive pomace as local biomass are important factors for the development of green processes targeting the valorization of this by-product into high-value molecules.

A Review on Application and Future prospects of Algae in Pharmaceutical and Food industry

Abstract: Algae are photosynthesis-producing organisms that can be found in freshwater, wastewater, and aquatic environments. In order to get around a number of high-tech barriers in the algae biomass sector, it is necessary to improve the various activities and research. Algae have the potential to supply novel chemicals and bioactive compounds for the biotechnology industry. The abundance of algal diversity must be utilized for various applications. Algal biomass is a source of energy (biofuels), fertilizer, pollution control, stabilization, nutrition, high-value molecules, and various bioactive metabolites that can be investigated for new drugs in terms of their applicability in local and global markets. Microalgae have been widely used for the production of biomass and biofuel. As a result, large-scale experimental setups have been built to produce a lot of biomass and biofuel. Food, cosmetics, pharmaceutical, and nutraceutical industries all benefit greatly from microalgae. They also produce numerous biomolecules with added value, such as polyunsaturated fatty acids, beta-1,3-glucan, astaxanthin, lutein, phycobiliprotein beta-carotene, and chlorophyll, in addition to the previously mentioned application. The pharmaceutical, cosmetic, food and feed, and nutraceutical industries all use these biomolecules extensively commercially. Furthermore, this review focuses specifically on the broad application potential algae based nonenergy applications, such as pharmaceuticals, food ingredients, pigments and cosmetics by marine algae.

Hollow Fiber Membrane for Organic Solvent Nanofiltration: A Mini Review.

Organic solvents take up 80% of the total chemicals used in pharmaceutical and related industries, while their reuse rate is less than 50%. Traditional solvent treatment methods such as distillation and evaporation have many disadvantages such as high cost, environmental unfriendliness, and difficulty in recovering heat-sensitive, high-value molecules. Organic solvent nanofiltration (OSN) has been a prevalent research topic for the separation and purification of organic solvent systems since the beginning of this century with the benefits of no-phase change, high operational flexibility, low cost, as well as environmental friendliness. Especially, hollow fiber (HF) OSN membranes have gained a lot of attention due to their high packing density and easy scale-up as compared with flat-sheet OSN membranes. This paper critically reviewed the recent research progress in the preparation of HF OSN membranes with high performance, including different materials, preparation methods, and modification treatments. This paper also predicts the future direction of HF OSN membrane development.

Functional diversity and metabolic engineering of plant-specialized metabolites

Abstract Plants are talented biochemists that produce a broad diversity of small molecules. These so-called specialized metabolites (SMs) play critical roles in the adaptive evolution of plants to defend against biotic and abiotic stresses, attract pollinators, and modulate soil microbiota for their own benefits. Many plant SMs have been used as nutrition and flavor compounds in our daily food, as well as drugs for treatment of human diseases. Current multi-omics tools have significantly accelerated the process of biosynthetic pathway elucidation in plants through correlation analyses, genetic mapping, and de novo biosynthetic gene cluster predictions. Understanding the biosynthesis of plant SMs has enabled reconstitution of naturally occurring specialized metabolic pathways in microbial hosts, providing a sustainable supply of these high-value molecules. In this review, we illustrate the general functions of several typical plant SMs in natural ecosystems and for human societies. We then provide an overview of current methods elucidating the biosynthetic pathways of plant SMs, and synthetic biology strategies that optimize the efficiency of heterologous biosynthetic pathways in microbial hosts. Moving forward, dissection of the functions and application of plant SMs by using current multidiscipline approaches would be greatly benefit to the scientific community and human societies.

Optimizing the Culture Medium of Lasiodiplodia sp. to Improve the Yield of Ethyl Acetate Extract as an Antimicrobial Source.

Endophytes often inhabit plant tissues and cause no disease symptoms. Lasiodiplodia is generally considered a pathogenic fungus, but such a genus is capable of producing high-value bioactive molecules, such as enzymes, secondary metabolites including antimicrobials. Therefore, Lasiodiplodia sp. endophyte was cultivated in static mode for 12days and EtOAc extracts were obtained and evaluated against pathogens afterward. Fermentation parameters (glucose, sucrose and NaNO3) were optimized by the factorial design and response surface methodology, as these are powerful tools to provide reliable information about fungal culture conditions and EtOAc extract yields were considered as response variables. Lasiodiplodia growth curve indicated that optimal production of EtOAc extract mass was achieved after 12days of fermentation (284mg 300mL-1 broth), which is in agreement with values obtained from validation tests. Minimum Inhibitory Concentration (MIC) and Minimum Microbicidal Concentration (MMC) essays suggested that the endophyte produce substances presenting antimicrobial and antifungal activities against ATCC Staphylococcus aureus and Candida albicans strains at optimum point under evaluated conditions. MIC values ranged between 50 and 100µgmL-1 for both pathogens, while MMC of C. albicans ranged from 100 to 200µgmL-1, which evidence its fungicidal effect. Furthermore, it was found that the EtOAc extract yield can be increased by optimizing carbon and nitrogen sources in endophyte cultivation, and there was good agreement between predicted and experimental values under optimized conditions. Thus, Lasiodiplodia fungi are promising sources of antimicrobials and changes in carbon and nitrogen sources can improve the yield of secondary metabolites according to the factorial design.

Jellyfish as an Alternative Source of Bioactive Antiproliferative Compounds.

Jellyfish are commonly considered a nuisance for their negative effects on human activities (e.g., fisheries, power plants and tourism) and human health. However, jellyfish provide several benefits to humans and are commonly eaten in eastern countries. Additionally, recent studies have suggested that jellyfish may become a source of high-value molecules. In this study, we tested the effects of the methanolic extracts and enriched fractions, obtained by solid-phase extraction fractionation, from the scyphomedusae Pelagia noctiluca, Rhizostoma pulmo, Cotylorhiza tuberculata and the cubomedusa Caryddea marsupialis on different human cancer cell lines in order to evaluate a potential antiproliferative activity. Our results indicated that fraction C from Caryddea marsupialis-(CM) and C. tuberculata oral arms (CTOA) were the most active to reduce cell viability in a dose-dependent manner. LC/MS based dereplication analyses highlighted that both bioactive fractions contained mainly fatty acids and derivatives, with CM additionally containing small peptides (0.7–0.8 kDa), which might contribute to its higher biological activity. The mechanism of action behind the most active fraction was investigated using PCR arrays. Results showed that the fraction C of CM can reduce the expression of genes involved in apoptosis inhibition in melanoma-treated cells, which makes jellyfish a potential new source of antiproliferative drugs to be exploited in the future.

Exploring the Potential of Lactic Acid Fermentation for the Recovery of Exhausted Vanilla Beans.

The market value of vanilla is constantly growing, as it is the aroma most appreciated by consumers worldwide. The key component of the aroma of vanilla beans is vanillin, which can be directly extracted from the plant, produced by chemical synthesis, or by bioconversion of natural precursors. Due to the increasing consumers' demand for products labeled as “natural,” extraction from vanilla pods results in a more valuable aroma source. Once the extraction is completed, what remains are the exhausted beans that still contain small seeds and other compounds, including varying amounts of vanillin trapped in the cellular structures of the plant. The application of fermentation of exhausted vanilla beans is proposed here as a strategy to recover “natural” vanillin and other valuable aroma compounds as a result of the metabolic conversion by lactic acid bacteria (LAB). The aim of this study was to verify the fermentability of exhausted vanilla beans by-products for their valorization, allowing the recovery of high-value molecules or new applications in food products. Design of Experiment (DoE) was used to screen a library of LAB strains to identify the best condition of fermentation in response to varying cultivation conditions. A comparison between mono and co-culture of LAB was assessed. Moreover, sensory panel tests and the evaluation of the aromatic components by Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry analysis were carried out to better understand the modification of the aroma profile after fermentation. Fermentation with LAB changed the volatile profile and sensory characteristics of the exhausted vanilla beans and represents a promising method for the valorization of these by-products.

Recombinant expression, purification and characterization of an active bacterial feruloyl-CoA synthase with potential for application in vanillin production

The ferulic acid (FA) represents a high-value molecule with applications in the cosmetic and pharmaceutical industries. This aromatic molecule is derived from lignin and can be enzymatically converted in other commercially interesting molecules, such as vanillin and bioplastics. This process starts with a common step of FA activation via CoA-thioesterification, catalyzed by feruloyl-CoA synthetases. Therefore, here, we report the successfully expression, purification as well as the initial structural and biochemical characterization of a stable, correctly folded, and catalytically active bacterial feruloyl-CoA synthase (here named FCS3) isolated from a lignin-degrading microbial consortium. The purification of recombinant FCS3 to near homogeneity was achieved using affinity chromatography. The FCS3 structure is composed of a mixture of α and β secondary structures and most likely forms stable homodimers in solution. The FCS3 presented a notable structural stability at alkaline pH values and it was able to convert FA and coenzyme A (CoA) into feruloyl-CoA complex at room temperature. This study should provide a useful basis for future biotechnological applications of FCS3, especially in the field of conversion of lignin-derived FA into high value compounds.

Applying biochemical and structural characterization of hydroxycinnamate catabolic enzymes from soil metagenome for lignin valorization strategies

The biocatalytic production of fuels and chemicals from plant biomass represents an attractive alternative to fossil fuel-based refineries. In this context, the mining and characterization of novel biocatalysts can promote disruptive innovation opportunities in the field of lignocellulose conversion and valorization. In the present work, we conducted the biochemical and structural characterization of two novel hydroxycinnamic acid catabolic enzymes, isolated from a lignin-degrading microbial consortium, a feruloyl-CoA synthetase, and a feruloyl-CoA hydratase-lyase, named LM-FCS2 and LM-FCHL2, respectively. Besides establishing the homology model structures for novel FCS and FCHL members with unique characteristics, the enzymes presented interesting biochemical features: LM-FCS2 showed stability in alkaline pHs and was able to convert a wide array of p-hydroxycinnamic acids to their respective CoA-thioesters, including sinapic acid; LM-FCHL2 efficiently converted feruloyl-CoA and p-coumaroyl-CoA into vanillin and 4-hydroxybenzaldehyde, respectively, and could produce vanillin directly from ferulic acid. The coupled reaction of LM-FCS2 and LM-FCHL2 produced vanillin, not only from commercial ferulic acid but also from a crude lignocellulosic hydrolysate. Collectively, this work illuminates the structure and function of two critical enzymes involved in converting ferulic acid into high-value molecules, thus providing valuable concepts applied to the development of plant biomass biorefineries. KEY POINTS: • Comprehensive characterization of feruloyl-CoA synthetase from metagenomic origin. • Novel low-resolution structures of hydroxycinnamate catabolic enzymes. • Production of vanillin via enzymatic reaction using lignocellulosic hydrolysates.

Advances in understanding the humins: Formation , prevention and application

The formation of dark-colored solids, as known as humins, was observed during acid-catalyzed dehydration of certain biomass platform molecules like fructose, glucose and 5-hydroxymethylfurfural (HMF). Humins structures were found to be precipitated as microspheres in the liquid phase and/or on the surfaces of solid catalyst and reactor walls. Humins formation begins with random and uncontrollable condensation of key intermediate molecules, namely, glucose, HMF, 1,2,4-Trihydroxybenzene (TB), 2,5-dioxo-6-hydroxyhexanal (DHH), etc. Further, mechanism network of humins formation also contains polymerization of soluble oligomers, growth of primary particles, physical absorption to soluble molecules and aggregation of particle groups. Humins formation retards the economy, safety and efficiency of cellulosic carbon conversion including wasting the substrates, blocking the reactor, causing deactivation of catalyst and absorbing the high-value molecules products. This necessitates the systematic elucidation of humins, including its formation mechanism network, characterization techniques and high-value utilization. This review organized significant studies of humins formation, including (i) background information of humins during different conversion process, (ii) different analytical techniques for humins characterization, (iii) the properties and formation mechanism of humins, (iv) prevention methods of humins formation and (v) exploration of high-value humins utilization.

Analysis of the stability of phycocyanin when trehalose and citric acid are used as protectants in nutraceutical gelatin candies under in vitro digestion assays’

Natural pigments are high value molecules for food industry as well as pharmacy, perceived as the best choice for today’s consumers given their origin and safety. However, its use is limited by its lability which hinders its application in products. In this work there was evaluated the stability of phycocyanin C in gelatin candies when added with trehalose and citric acid; the formulated candies were digested in synthetic gastric juice with pepsin. There were calculated predictive equations correlating the concentration of pigment as function of time, additives, and enzymatic presence. It was observed that phycocyanin can supports the gastric juice and pepsin when combined with trehalose for enough time to pass thru the digestive tract. It was concluded that the inclusion of trehalose in the formulation led to a longer stability of the pigment. Besides, it was determined that gelatin candies are a good vector to deliver phycocyanin C to the intestine.

Prospective on the application of abiotic stresses to enhance the industrial production of exopolysaccharides from microalgae

Microalgae species are sustainable sources for diverse bioactive compounds of commercial interest. Within the framework of bioeconomy, exopolysaccharides (EPS) from microalgal sources stand out as potential high-value molecules in the cosmetic, nutraceutical, and pharmaceutical industries. Since the goal is to increase the EPS presence in those markets, prospecting potential applications through the characterization of species–structure–bioactivity relations and optimizing EPS production yields through sustainable and economically feasible strategies are needed. This graphical review addresses current strategies aimed to increase EPS production in microalgal species to reach industrial-scale production with a particular focus on the use of abiotic stress – a strategy that adversely affects cell growth performance but induces desirable changes in the metabolic profile of the microalgae requiring minor to mild modifications in already established production settings. Therefore, analysis of the stress-based approaches to increase EPS production at an industrial scale is discussed, identifying those factors with the most potential from technological and economic perspectives.

Microbial synthesis of wax esters

Microbial synthesis of wax esters (WE) from low-cost renewable and sustainable feedstocks is a promising path to achieve cost-effectiveness in biomanufacturing. WE are industrially high-value molecules, which are widely used for applications in chemical, pharmaceutical, and food industries. Since the natural WE resources are limited, the WE production mostly rely on chemical synthesis from rather expensive starting materials, and therefore solution are sought from development of efficient microbial cell factories. Here we report to engineer the yeast Yarrowia lipolytica and bacterium Escherichia coli to produce WE at the highest level up to date. First, the key genes encoding fatty acyl-CoA reductases and wax ester synthase from different sources were investigated, and the expression system for two different Y. lipolytica hosts were compared and optimized for enhanced WE production and the strain stability. To improve the metabolic pathway efficiency, different carbon sources including glucose, free fatty acid, soybean oil, and waste cooking oil (WCO) were compared, and the corresponding pathway engineering strategies were optimized. It was found that using a lipid substrate such as WCO to replace glucose led to a 60-fold increase in WE production. The engineered yeast was able to produce 7.6 g/L WE with a yield of 0.31 (g/g) from WCO within 120 h and the produced WE contributed to 57% of the yeast DCW. After that, E. coli BL21(DE3), with a faster growth rate than the yeast, was engineered to significantly improve the WE production rate. Optimization of the expression system and the substrate feeding strategies led to production of 3.7–4.0 g/L WE within 40 h in a 1-L bioreactor. The predominant intracellular WE produced by both Y. lipolytica and E. coli in the presence of hydrophobic substrates as sole carbon sources were C36, C34 and C32, in an order of decreasing abundance and with a large proportion being unsaturated. This work paved the way for the biomanufacturing of WE at a large scale.

Alkyne Surrogates in Cycloaddition Reactions for the Preparation of Molecules of Interest

AbstractAlthough alkynes are very good partners for cycloaddition reactions, their use is often associated to several drawbacks, such as multi‐steps synthesis, selectivity and stability issues or difficulty to handle for alkynes with low boiling point. In this context, alkyne surrogates have emerged as alternative substitution partners unlocking new opportunities. This review intends to highlight the outstanding power and utility of such compounds for the preparation of high value molecules.

Production of xylitol and carotenoids from switchgrass and Eucalyptus globulus hydrolysates obtained by intensified steam explosion pretreatment

This study investigated the fermentability of hemicellulosic hydrolysates obtained by intensified steam explosion pretreatment of switchgrass (Panicum virgatum) and Eucalyptus globulus. Xylitol and carotenoids were the high-value molecules produced by fermentation. The intensified pretreatment allowed to process a large amount of biomass per unit of time and resulted in hydrolysates containing high amount of sugars, among of which, a significant fraction was in the form of oligomers (eucalyptus hydrolysate: 25.0 g/L of oligomers and 50.6 g/L of monomeric sugars; switchgrass hydrolysate: 18.9 g/L of oligomers and 39.6 g/L of monomeric sugars). To be used as fermentation media, a post-hydrolysis step was applied to increase the content of monomeric sugars in the hydrolysates. Then, a detoxification process was carried out to reduce the concentration of inhibitors present. Two evolved yeasts were used for fermentation: Kluyveromyces marxianus for xylitol production, and Rhodosporidium toruloides for carotenoids production. Results revealed that the hydrolysates produced by intensified steam explosion of switchgrass and eucalyptus present good fermentability and can be used to produce valuable compounds such as xylitol, after detoxification. K. marxianus presented better tolerance to inhibitory compounds still present in the detoxified hydrolysates (acetic acid up to 3.94 g/L and phenolic compounds up to 2.28 g/L) than R. toruloides, which favored the production of xylitol. Finally, the intensified pretreatment was found to be a potential strategy to obtain hydrolysates with high concentration of sugars, reducing the need of concentration in a subsequent step. Moreover, the detoxification strategy applied in this study allowed to recover valuable compounds from the hydrolysates, offering extra value to a biorefinery. Altogether, the findings of this study contribute to the advancement of a technology for valorization of hemicellulosic hydrolysates.

Valorisation of Brewer’s Spent Yeasts’ Hydrolysates as High-Value Bioactive Molecules

Brewer’s spent yeast (BSY) is produced by the beer industry and has high nutritional value and great potential for producing high-value molecules, such as peptides, for nutraceutical, food and feed applications. In the present research, Flavourzyme® and Protamex® enzymes were selected for protein hydrolysis based on previous studies. The optimum conditions for the enzymatic hydrolysis were defined by response surface methodology (RSM) by the Box–Behnken design composed of four variables: temperature, pH, enzyme dosage and time. Protein content, hydrolysis degree and the anti-microbial and antioxidant bioactivities of obtained hydrolysates were quantified. Obtained results show that time, enzyme dosage and pH had the highest effect on protein extraction yield (PEY), degree of hydrolysis (DH) and antioxidant activity. Response variables ranged from 13.7 to 29.7% for PEY, from 6.3 to 35.7% for DH and from 0.65 to 1.65 g for Trolox equivalent antioxidant capacity. Antimicrobial activity, measured as minimum inhibitory concentration, against Aeromonas salmonicida, Bacillus cereus, Bacillus subtilis and Salmonella enterica, ranged from 6.25 to 50 mg/mL. Antioxidant and antimicrobial activity showed the potential use of BSY hydrolysates as an ingredient for functional foods.

Induction of biosynthesis of ketocarotenoid from β-carotene in fish embryos

Ketocarotenoids are high-value molecules with powerful antioxidant properties. They are widely used as a feed supplements due to their vital functions for animals, particularly for fish and marine animals. However, fish is not able to synthesize ketocarotenoids but consume them from diet. Here, we report metabolic engineering of fish cells and fish embryos for biosynthesis of ketocarotenoid from β-carotene as a substrate. Firstly, we introduced two sets of fish-codon optimized genes (crtW and crtZ) from Brevundimonas sp., and (crtS and crtR) from Xanthophyllomyces dendrorhous, to the cultured common carp cells (Epithelioma papulosum cyprinid, EPC cells) to test the production of ketocarotenoids in vitro. We then introduced the optimized set of genes, crtW and crtZ, to zebrafish (Danio rerio) embryos and found that they could efficiently mediate the synthesis of ketocarotenoids from β-carotene in vivo. Subsequently, we constructed a bicistronic expression vector containing crtW and crtZ genes linked with a P2A peptide coding sequence, and evaluated the production of ketocarotenoids using both in vitro and in vivo systems. Overall, we unprecedentedly stimulated fish cells and zebrafish embryos to synthesize ketocarotenoids with a total content of 29.9 μg/g DW and 16.46 μg/g DW, respectively. Furthermore, we successfully optimized the ketocarotenoids biosynthesis and significantly increased the total ketocarotenoids content in fish models up to 2.5 and 2.14 fold, corresponding to 75.24 and 35.25 μg/g DW, respectively. This study is a proof of concept of engineering animals to manufacture ketocarotenoids and provides a pilot study to enable fish to synthesize ketocarotenoids in vivo.