Protein and lysine improvement harnessed by a signal chain of red light-emitting diode light in Chlorella pyrenoidosa.

Microalgae are emerging as a novel single-cell protein source that can substitute traditional plant protein feeds. In this investigation, lysine and protein accumulation in Chlorella pyrenoidosa were significantly enhanced under red light-emitting diode light, addressing challenge of limiting amino acid in plant proteins. The study employed targeted metabolomics, HPLC, and qRT-PCR to validate the light-induced pathway triggering lysine biosynthesis. Specifically, the pathway involves Ca2+-CaM as an intermediary in signal transduction, which directly inhibits PEPC activity. This inhibition directs a significant carbon flux towards central carbon metabolism, resulting in increased pyruvate levels-a critical precursor for lysine biosynthesis via the diaminopimelate pathway. Ultimately, the content of protein and lysine under red light increased by 36.02 % and 99.56 %, respectively, compared to those under white light. These findings provide a novel orientation for the precise regulation of lysine accumulation in microalgae, and moreover lay a solid theoretical foundation for producing microalgal proteins.

High-efficiency expression of alginate lyase in Pichia pastoris facilitated by Vitreoscilla hemoglobin.

Vitreoscilla hemoglobin (VHb) can enhance the ability of recombinant strains to express heterologous proteins under low-oxygen conditions. However, its mechanism of action in the Pichia pastoris expression system remains unclear. In this study, three VHb construction strategies were designed to elucidate the mechanisms by which VHb promotes heterologous protein expression in P. pastoris. Notably, the co-expression pattern involving the sequential expression of the 102C300C gene followed by the Vgb gene significantly improved enzyme activity in the recombinant strain X33-102C300C-Vgb. The enzyme activity was 203.4 ±â€¯0.57 U/mL at 180 h of fermentation in the 5-L system, which was 20.7 % higher than that of the starting strain X33-102C300C. Fluorescent labeling experiments revealed for the first time that a dual-transcription unit approach achieved superior VHb expression, indicating its potential for further development. Furthermore, transcriptomic and metabolomic analyses demonstrated that VHb enhances the growth of recombinant yeast colonies by improving respiration-related metabolism under low-oxygen conditions. This, in turn, alleviated the repression of the expression alcohol oxidase (AOX) at high methanol concentrations, resulting in increased alginate lyase activity. This study provides a theoretical foundation for improving the target protein expression in recombinant P. pastoris during high-density fermentation.

Identification, expression, and characterization of a marine-derived chitinase Ce0303 from Chitiniphilus eburneus YS-30 with exo- and endo-hydrolytic properties.

N-acetyl-oligosaccharides exhibit antioxidant and antibacterial activities. However, the low catalytic efficiency of chitinase on crystalline chitin hinders the eco-friendly production of N-acetyl-oligosaccharides. A marine-derived chitinase-producing strain Chitiniphilus eburneus YS-30 was screened in this study. The genome of C. eburneus YS-30 spans 4,522,240 bp, with a G + C content of 63.96 % and 4244 coding genes. Among the chitinases secreted by C. eburneus YS-30, Ce0303 showed the highest content at 19.10 %, with a molecular weight of 73.5 kDa. Recombinant Ce0303 exhibited optimal activity at 50 °C and pH 5.0, maintaining stability across pH 4.0-10.0. Ce0303 demonstrated strict substrate specificity, with a specific activity toward colloidal chitin of 6.41 U mg-1, Km of 2.34 mg mL-1, and kcat of 3.27 s-1. The specific activity of Ce0303 toward α-chitin was 18.87 % of its activity on colloidal chitin. Ce0303 displayed both exo- and endo-hydrolytic properties, primarily producing (GlcNAc)1-3 from colloidal chitin. The structure of Ce0303 includes one catalytic domain and two chitin-binding domains. Docking results revealed that the GlcNAc at -1 subsite formed two hydrogen bonds with conserved Trp380. The hydrolytic properties of Ce0303 will provide technical support for the comprehensive utilization of crustacean raw materials.

Phycocyanin from microalgae: A comprehensive review covering microalgal culture, phycocyanin sources and stability.

As food safety continues to gain prominence, phycocyanin (PC) is increasingly favored by consumers as a natural blue pigment, which is extracted from microalgae and serves the dual function of promoting health and providing coloration. Spirulina-derived PC demonstrates exceptional stability within temperature ranges below 45 °C and under pH conditions between 5.5 and 6.0. However, its application is limited in scenarios involving high-temperature processing due to its sensitivity to heat and light. This comprehensive review provides insights into the efficient production of PC from microalgae, covers the metabolic engineering of microalgae to increase PC yields and discusses various strategies for enhancing its stability in food applications. In addition to the most widely used Spirulina, some red algae and Thermosynechococcus can serve as good source of PC. The genetic and metabolic manipulation of microalgae strains has shown promise in increasing PC yield and improving its quality. Delivery systems including nanoparticles, hydrogels, emulsions, and microcapsules offer a promising solution to protect and extend the shelf life of PC in food products, ensuring its vibrant color and health-promoting properties are preserved. This review highlights the importance of metabolic engineering, multi-omics applications, and innovative delivery systems in unlocking the full potential of this natural blue pigment in the realm of food applications, provides a complete overview of the entire process from production to commercialization of PC, including the extraction and purification.

Development strategies and application of antimicrobial peptides as future alternatives to in-feed antibiotics.

The use of in-feed antibiotics has been widely restricted due to the significant environmental pollution and food safety concerns they have caused. Antimicrobial peptides (AMPs) have attracted widespread attention as potential future alternatives to in-feed antibiotics owing to their demonstrated antimicrobial activity and environment friendly characteristics. However, the challenges of weak bioactivity, immature stability, and low production yields of natural AMPs impede practical application in the feed industry. To address these problems, efforts have been made to develop strategies for approaching the AMPs with enhanced properties. Herein, we summarize approaches to improving the properties of AMPs as potential alternatives to in-feed antibiotics, mainly including optimization of structural parameters, sequence modification, selection of microbial hosts, fusion expression, and industrially fermentation control. Additionally, the potential for application of AMPs in animal husbandry is discussed. This comprehensive review lays a strong theoretical foundation for the development of in-feed AMPs to achieve the public health globally.

Computer-Aided Rational Design Strategy to Improve the Thermal Stability of Alginate Lyase AlyMc.

Alginate lyase degrades alginate by the ß-elimination mechanism to produce unsaturated alginate oligosaccharides (UAOS), which have better bioactivities than saturated AOS. Enhancing the thermal stability of alginate lyases is crucial for their industrial applications. In this study, a feasible and efficient rational design strategy was proposed by combining the computer-aided ΔΔG value calculation with the B-factor analysis. Two thermal stability-enhanced mutants, Q246V and K249V, were obtained by site-directed mutagenesis. Particularly, the t1/2, 50 °C for mutants Q246V and K249V was increased from 2.36 to 3.85 and 3.65 h, respectively. Remarkably, the specific activities of Q246V and K249V were enhanced to 2.41- and 2.96-fold that of alginate lyase AlyMc, respectively. Structural analysis and molecular dynamics simulations suggested that mutations enhanced the hydrogen bond networks and the overall rigidity of the molecular structure. Notably, mutant Q246V exhibited excellent thermal stability among the PL-7 alginate lyase family, especially considering the heightened enzymatic activity. Moreover, the rational design strategy used in this study can effectively improve the thermal stability of enzymes and has important significance in advancing applications of alginate lyase.

Selecting a preculture strategy for improving biomass and astaxanthin productivity of Chromochloris zofingiensis.

Chromochloris zofingiensis is a potential source of natural astaxanthin; however, its rapid growth and astaxanthin enrichment cannot be achieved simultaneously. This study established autotrophic, mixotrophic, and heterotrophic preculture patterns to assess their ameliorative effect on the C. zofingiensis heterotrophic growth state. In comparison, mixotrophic preculture (MP) exhibited the best improving effect on heterotrophic biomass concentration of C. zofingiensis (up to 121.5 g L-1) in a 20 L fermenter, reaching the global leading level. The astaxanthin productivity achieved 111 mg L-1 day-1, 7.4-fold higher than the best record. The transcriptome and 13C tracer-based metabolic flux analysis were used for mechanism inquiry. The results revealed that MP promoted carotenoid and lipid synthesis, and supported synthesis preference of low unsaturated fatty acids represented by C18:1 and C16:0. The MP group maintained the best astaxanthin productivity via mastering the balance between increasing glucose metabolism and inhibition of carotenoid synthesis. The MP strategy optimized the physiological state of C. zofingiensis and realized its heterotrophic high-density growth for an excellent astaxanthin yield on a pilot scale. This strategy exhibits great application potential in the microalgae-related industry. KEY POINTS: • Preculture strategies changed carbon flux and gene expression in C. zofingiensis • C. zofingiensis realized a high-density culture with MP and fed-batch culture (FBC) • Astaxanthin productivity achieved 0.111 g L-1 day-1 with MP and FBC.

Realization process of microalgal biorefinery: The optional approach toward carbon net-zero emission.

Increasing carbon dioxide (CO2) emission has already become a dire threat to the human race and Earth's ecology. Microalgae are recommended to be engineered as CO2 fixers in biorefinery, which play crucial roles in responding climate change and accelerating the transition to a sustainable future. This review sorted through each segment of microalgal biorefinery to explore the potential for its practical implementation and commercialization, offering valuable insights into research trends and identifies challenges that needed to be addressed in the development process. Firstly, the known mechanisms of microalgal photosynthetic CO2 fixation and the approaches for strain improvement were summarized. The significance of process regulation for strengthening fixation efficiency and augmenting competitiveness was emphasized, with a specific focus on CO2 and light optimization strategies. Thereafter, the massive potential of microalgal refineries for various bioresource production was discussed in detail, and the integration with contaminant reclamation was mentioned for economic and ecological benefits. Subsequently, economic and environmental impacts of microalgal biorefinery were evaluated via life cycle assessment (LCA) and techno-economic analysis (TEA) to lit up commercial feasibility. Finally, the current obstacles and future perspectives were discussed objectively to offer an impartial reference for future researchers and investors.

Potential Application of Marine Fucosyl-Polysaccharides in Regulating Blood Glucose and Hyperglycemic Complications.

Diabetes mellitus (DM) has become the world's third major disease after tumors and cardiovascular disease. With the exploitation of marine biological resources, the efficacy of using polysaccharides isolated from marine organisms in blood glucose regulation has received widespread attention. Some marine polysaccharides can reduce blood glucose by inhibiting digestive enzyme activity, eliminating insulin resistance, and regulating gut microbiota. These polysaccharides are mainly fucose-containing sulphated polysaccharides from algae and sea cucumbers. It follows that the hypoglycemic activity of marine fucosyl-polysaccharides is closely related to their structure, such as their sulfate group, monosaccharide composition, molecular weight and glycosidic bond type. However, the structure of marine fucosyl-polysaccharides and the mechanism of their hypoglycemic activity are not yet clear. Therefore, this review comprehensively covers the effects of marine fucosyl-polysaccharides sources, mechanisms and the structure-activity relationship on hypoglycemic activity. Moreover, the potential regulatory effects of fucosyl-polysaccharides on vascular complications caused by hyperglycemia are also summarized in this review. This review provides rationales for the activity study of marine fucosyl-polysaccharides and new insights into the high-value utilization of marine biological resources.

Response of Salmonella enterica serovar Typhimurium to alginate oligosaccharides fermented with fecal inoculum: integrated transcriptomic and metabolomic analyses.

Alginate oligosaccharides (AOS), extracted from marine brown algae, are a common functional feed additive; however, it remains unclear whether they modulate the gut microbiota and microbial metabolites. The response of Salmonella enterica serovar Typhimurium, a common poultry pathogen, to AOS fermented with chicken fecal inocula was investigated using metabolomic and transcriptomic analyses. Single-strain cultivation tests showed that AOS did not directly inhibit the growth of S. Typhimurium. However, when AOS were fermented by chicken fecal microbiota, the supernatant of fermented AOS (F-AOS) exhibited remarkable antibacterial activity against S. Typhimurium, decreasing the abundance ratio of S. Typhimurium in the fecal microbiota from 18.94 to 2.94%. Transcriptomic analyses showed that the 855 differentially expressed genes induced by F-AOS were mainly enriched in porphyrin and chlorophyll metabolism, oxidative phosphorylation, and Salmonella infection-related pathways. RT-qPCR confirmed that F-AOS downregulated key genes involved in flagellar assembly and the type III secretory system of S. Typhimurium, indicating metabolites in F-AOS can influence the growth and metabolism of S. Typhimurium. Metabolomic analyses showed that 205 microbial metabolites were significantly altered in F-AOS. Among them, the increase in indolelactic acid and 3-indolepropionic acid levels were further confirmed using HPLC. This study provides a new perspective for the application of AOS as a feed additive against pathogenic intestinal bacteria. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00176-z.

Structural characterization of a novel fucosylated trisaccharide prepared from bacterial exopolysaccharides and evaluation of its prebiotic activity.

Fucosylated oligosaccharides have promising prospects in various fields. In this study, a fucosylated trisaccharide (GFG) was separated from the acidolysis products of exopolysaccharides from Clavibacter michiganensis M1. Structural characterization demonstrated that GFG consists of glucose, galactose, and fucose, with a molecular weight of 488 Da. Nuclear magnetic resonance analysis showed that it has a different structure than that of 2'-fucosyllactose (2'-FL), even though they have the same monosaccharide composition. In vitro prebiotic experiments were conducted to evaluate the differences in the utilization of three selected carbohydrates by fourteen bacterial strains. In comparison with 2'-FL, GFG could be utilized by more beneficial bacteria, leading to generate more short-chain fatty acids. Moreover, GFG could not promote the proliferation of Escherichia coli. This work describes a novel fucosylated oligosaccharide and its preparation method, and the obtained trisaccharide may serve as a promising candidate for fucosylated human milk oligosaccharides.

High-Efficiency Fermentation of Nattokinase by Recombinant PSP2 Using Oyster Protein Hydrolysate as a Substrate.

In recent years, cardiovascular and cerebrovascular diseases have been the focus of several studies. In this study, oyster protein hydrolysate was produced via enzyme hydrolysis and used as a fermentation substrate to ferment recombinant strain PSP2 to produce nattokinase. Using the synergism strategy, fermentation products with fibrinolytic and angiotensin I-converting enzyme (ACE) inhibitory activities were obtained and evaluated. The fermentation medium contained 1.0% trypsin, 1.0% oyster protein hydrolysate, 2.0% maltose, and 0.5% sodium chloride, with an initial pH of 7.0. The maximum nattokinase activity was 390.23 ± 10.24 FU/mL after 72 h of fermentation. The flavor of the product was improved, and heavy metals and volatile salt nitrogen were partially removed via fermentation. The ACE inhibitory activity (IC50) of the fermentation products was 1.433 mg/mL. This study provides a novel approach for the development of marine functional foods with hypotensive and antithrombotic properties.

Technological readiness of commercial microalgae species for foods.

Microalgae have great potential as a future source to meet the increasing global demand for foods. Several microalgae are permitted as safety sources in different countries and regions, and processed as commercial products. However, edible safety, economic feasibility, and acceptable taste are the main challenges for microalgal application in the food industry. Overcome such challenges by developing technology accelerates transition of microalgae into sustainable and nutritious diets. In this review, edible safety of Spirulina, Chlamydomonas reinhardtii, Chlorella, Haematococcus pluvialis, Dunaliella salina, Schizochytrium and Nannochloropsis is introduced, and health benefits of microalgae-derived carotenoids, amino acids, and fatty acids are discussed. Technologies of adaptive laboratory evolution, kinetic model, bioreactor design and genetic engineering are proposed to improve the organoleptic traits and economic feasibility of microalgae. Then, current technologies of decoloration and de-fishy are summarized to provide options for processing. Novel technologies of extrusion cooking, delivery systems, and 3D bioprinting are suggested to improve food quality. The production costs, biomass values, and markets of microalgal products are analyzed to reveal the economic feasibility of microalgal production. Finally, challenges and future perspectives are proposed. Social acceptance is the major limitation of microalgae-derived foods, and further efforts are required toward the improvement of processing technology.

Clostridium butyricum Prevents Dysbiosis and the Rise in Blood Pressure in Spontaneously Hypertensive Rats.

Hypertension is accompanied by dysbiosis and a decrease in the relative abundance of short-chain fatty acid (SCFA)-producing bacteria. However, there is no report to examine the role of C. butyricum in blood pressure regulation. We hypothesized that a decrease in the relative abundance of SCFA-producing bacteria in the gut was the cause of spontaneously hypertensive rats (SHR)-induced hypertension. C. butyricum and captopril were used to treat adult SHR for six weeks. C. butyricum modulated SHR-induced dysbiosis and significantly reduced systolic blood pressure (SBP) in SHR (p < 0.01). A 16S rRNA analysis determined changes in the relative abundance of the mainly SCFA-producing bacteria Akkermansia muciniphila, Lactobacillus amylovorus, and Agthobacter rectalis, which increased significantly. Total SCFAs, and particularly butyrate concentrations, in the SHR cecum and plasma were reduced (p < 0.05), while C. butyricum prevented this effect. Likewise, we supplemented SHR with butyrate for six weeks. We analyzed the flora composition, cecum SCFA concentration, and inflammatory response. The results showed that butyrate prevented SHR-induced hypertension and inflammation, and the decline of cecum SCFA concentrations (p < 0.05). This research revealed that increasing cecum butyrate concentrations by probiotics, or direct butyrate supplementation, prevented the adverse effects of SHR on intestinal flora, vascular, and blood pressure.

Stimulating carbon and nitrogen metabolism of Chlorella pyrenoidosa to treat aquaculture wastewater and produce high-quality protein in plate photobioreactors.

Wastewater treatment by microalgae is the economical and environmentally friendly strategy, but is still challenged with the strict discharge standards and valuable biomass exploitations. The carbon and nitrogen metabolism of Chlorella pyrenoidosa was improved by the red LED light and starch addition to treat Tilapia aquaculture wastewater (T-AW) and produce protein simultaneously in a plate photobioreactor. The red LED light was applied to improve the nutrient removals at an outdoor temperature, but the concentrations except total nitrogen did not satisfy the discharge standards. After starch addition, the removal efficiencies of total phosphorus, total nitrogen, chemical oxygen demand, and total ammonia nitrogen were 85.15, 96.96, 88.53, and 98.01 % in a flat-plate photobioreactor, respectively, which met the discharge standards and the protein production reached 0.60 g/L. At a molecular level, the metabolic flux and transcriptome analyses showed that red light promoted carbon flux of the Embden-Meyerhof-Pranas pathway and tricarboxylic cycle, and upregulated the levels of genes encoding α-amylase, glutamine synthetase, glutamate dehydrogenase, nitrate transporter, and ammonium transporter, which facilitated nutrients removal and provided nitrogen sources for protein biosynthesis. The harvesting C. pyrenoidosa possessed the 62 % essential amino acids and great lipid composition for biofuels. This study provided a new orientation for outdoor wastewater treatment and protein production by collaboratively regulating the carbon and nitrogen metabolism of microalgae.

Microalgae-Derived Pigments for the Food Industry.

In the food industry, manufacturers and customers have paid more attention to natural pigments instead of the synthetic counterparts for their excellent coloring ability and healthy properties. Microalgae are proven as one of the major photosynthesizers of naturally derived commercial pigments, gaining higher value in the global food pigment market. Microalgae-derived pigments, especially chlorophylls, carotenoids and phycobiliproteins, have unique colors and molecular structures, respectively, and show different physiological activities and health effects in the human body. This review provides recent updates on characteristics, application fields, stability in production and extraction processes of chlorophylls, carotenoids and phycobiliproteins to standardize and analyze their commercial production from microalgae. Potential food commodities for the pigment as eco-friendly colorants, nutraceuticals, and antioxidants are summarized for the target products. Then, recent cultivation strategies, metabolic and genomic designs are presented for high pigment productivity. Technical bottlenecks of downstream processing are discussed for improved stability and bioaccessibility during production. The production strategies of microalgal pigments have been exploited to varying degrees, with some already being applied at scale while others remain at the laboratory level. Finally, some factors affecting their global market value and future prospects are proposed. The microalgae-derived pigments have great potential in the food industry due to their high nutritional value and competitive production cost.

Butyrogenic effect of galactosyl and mannosyl carbohydrates and their regulation on piglet intestinal microbiota.

Diarrhea is a global problem that causes economic losses in the pig industry. There is a growing attention on finding new alternatives to antibiotics to solve this problem. Hence, this study aimed to compare the prebiotic activity of low-molecular-weight hydrolyzed guar gum (GMPS) with commercial manno-oligosaccharide (MOS) and galacto-oligosaccharide (GOS). We further identified their combined effects along with probiotic Clostridium butyricum on regulating the intestinal microbiota of diarrheal piglet by in vitro fermentation. All the tested non-digestible carbohydrates (NDCs) showed favorable short-chain fatty acid-producing activity, and GOS and GMPS showed the highest production of lactate and butyrate, respectively. After 48 h of fermentation, the greatest enhancement in the abundance of Clostridium sensu stricto 1 was observed with the combination of GMPS and C. butyricum. Notably, all the selected NDCs significantly decreased the abundances of pathogenic bacteria genera Escherichia-Shigella and Fusobacterium and reduced the production of potentially toxic metabolites, including ammonia nitrogen, indole, and skatole. These findings demonstrated that by associating with the chemical structure, GMPS exhibited butyrogenic effects in stimulating the proliferation of C. butyricum. Thus, our results provided a theoretical foundation for further application of galactosyl and mannosyl NDCs in the livestock industry. KEY POINTS: • Galactosyl and mannosyl NDCs showed selective prebiotic effects. • GMPS, GOS, and MOS reduced pathogenic bacteria and toxic metabolites production. • GMPS specifically enhanced the Clostridium sensu stricto 1 and butyrate production.

Altering autotrophic carbon metabolism of Nitzschia closterium to mixotrophic mode for high-value product improvement.

An adaptive laboratory evolution (ALE) strategy was designed to evolve autotrophic Nitzschia closterium to mixotrophic growth for high productivity of essential amino acid (EAA), eicosapentaenoic acid (EPA) and fucoxanthin. The N. closterium growth was limited under glucose initially, but a red light emitting diode was innovatively applied to modify carbon metabolism and obtain mixotrophic strain of N. closterium GM. The N. closterium GM biomass concentration was improved by 65.07% comparing with wild type, but exhibited weak photosynthesis and strong glucose metabolism. At carbon metabolism levels, ALE promoted NADPH oxidase activity and induced protein degradation to lipid biosynthesis by elevating acetyl-CoA and pyruvate contents. It also improved carbon flux to TCA cycle, and elevated contents of glucose-6-phosphate, fructose-6-phosphate, glyceraldehyde-3-phosphate for providing sufficient ATP and NADPH. Productivities of EPA, EAA and fucoxanthin were increased by 41.0%, 18.8% and 20.4%, respectively. This ALE strategy was promising in microalgal production of high-value products.

Fucoxanthin from marine microalgae: A promising bioactive compound for industrial production and food application.

Fucoxanthin attracts increasing attentions due to its potential health benefits, which has been exploited in several food commodities. However, fucoxanthin available for industrial application is mainly derived from macroalgae, and is not yet sufficiently cost-effective compared with microalgae. This review focuses on the strategies to improve fucoxanthin productivity and approaches to reduce downstream costs in microalgal production. Here we comprehensively and critically discuss ways and methods to increase the cell growth rate and fucoxanthin content of marine microalgae, including strain screening, condition optimization, design of culture mode, metabolic and genetic engineering, and scale-up production of fucoxanthin. The approaches in downstream processes provide promising alternatives for fucoxanthin production from marine microalgae. Besides, this review summarizes fucoxanthin improvements in solubility and bioavailability by delivery system of emulsion, nanoparticle, and hydrogel, and discusses fucoxanthin metabolism with gut microbes. Fucoxanthin production from marine microalgae possesses numerous advantages in environmental sustainability and final profits to meet incremental global market demands of fucoxanthin. Strategies of adaptive evolution, multi-stage cultivation, and bioreactor improvements have tremendous potentials to improve economic viability of the production. Moreover, fucoxanthin is promising as the microbiota-targeted ingredient, and nanoparticles can protect fucoxanthin from external environmental factors for improving the solubility and bioavailability.