The diamondback moth (Plutella xylostella) is a devastating global pest of cruciferous crops. This study explores the potential of targeting the vacuolar ATPase subunit E (PxvATPaseE) for its control. We demonstrate that PxvATPaseE is essential for larval development, showing high expression levels in the midgut. RNA interference (RNAi)-mediated silencing of PxvATPaseE resulted in severe growth retardation and dose-dependent mortality, with higher dsRNA doses inducing more sustained effects. Furthermore, computational virtual screening of natural compound libraries identified a high-affinity binder of PxvATPaseE, such as periplocoside D. Our results demonstrate that PxvATPaseE is a promising molecular target for controlling P. xylostella, supporting a dual-strategy approach combining RNAi and targeted chemical inhibition for future pest management solutions.

Omega-3 polyunsaturated fatty acids (PUFAs) are essential for human health due to their numerous beneficial biological properties. These compounds are synthesized in marine bacteria and eukaryotic microalgae by PUFA megasynthases (Pfas), which are evolutionarily related to fatty acid synthases (FAS) and polyketide synthases (PKS). In FAS, PKS, and PUFA synthases, the acyltransferase (AT) domain plays a critical role in condensation reactions by loading starter or extender units into the acyl carrier protein (ACP) domain. PfaB, a component of PUFA megasynthases, harbors a pseudo-ketosynthase (KS') domain and an AT domain. In this study, we show that PfaB determines the final PUFA product, as demonstrated by in vivo assays in Escherichia coli using the DHA-producing Moritella marina and the EPA-producing Shewanella baltica. In vitro biochemical assays confirm that PfaB exhibits acyltransferase activity, with distinct substrate specificity from the AT domain of PfaA. Finally, we report the crystal structure of PfaB from S. baltica, representing the first structurally resolved AT domain within a PUFA megasynthase. Molecular docking analyses suggest that specific residues may contribute to differences in substrate recognition and specificity. Together, these findings show that PfaB acts as the terminal acyltransferase, providing new insights into its functional role in PUFA biosynthesis, and advancing our understanding of its mechanism and ligand interactions.

Lipid droplets (LDs) are dynamic organelles essential for lipid and energy homeostasis. Despite their presence in marine thraustochytrids, little is understood about the protein composition and biological functions of LDs from this source. We investigated the lipid profile of LDs extracted from a docosahexaenoic acid (DHA)-producing Schizochytrium sp. strain R1. Our analysis revealed the accumulation of DHA-enriched neutral lipids and phospholipids within LDs. Proteomic analysis of isolated LDs identified hundreds of proteins involved in LD dynamics, lipid metabolism, protein synthesis, and degradation. Notably, we discovered a scaffold protein, ScLDAP, which was highly concentrated in LDs and shared sequence homology with a thraustochytrid-specific lipid droplet protein but not with any LD proteins in bacteria, animals, or plants. Overexpressing Scldap in Yarrowia lipolytica resulted in a 3.7-fold increase in total lipids and more LDs, while its overexpression in Schizochytrium sp. strain M326 led to an 88.1 % increase in TFA and an 89.5 % increase in DHA titer. However, no synergistic effects were observed when co-expressed with the acyl-CoA: diacylglycerol acyltransferase gene from Schizochytrium sp.. Additionally, two auxiliary LD proteins with unknown functions were found to contribute to the increased total lipids. This study elucidates the complexity of LDs in Schizochytrium sp. and highlights the potential applications of LD-associated proteins in lipid production.

In this study, four rapeseed protein extracts were prepared from intact/dehulled rapeseed using salt and alkaline extraction. Their nutritional quality was assessed and linked to protein composition through 4D label-free proteomics. Salt-extracted protein isolates had lower glucosinolate (0.47 μmol/g) and phytic acid (0.59 %) content than the alkaline-extracted protein isolates, whereas the alkaline-extracted isolates demonstrated higher digestibility (93 %). The inconsistency between the trend of non-protein anti-nutritional factors and protein digestibility suggests that protein composition may play a more significant role than anti-nutritional factors. Proteomic analysis showed that both extraction methods yielded similar globulin (cruciferin) abundance, but the salt-extracted isolate contained more protein anti-nutritional factors, including albumin (napin) and other protease inhibitors. These findings highlight the importance of protein composition in determining digestibility, showing the need for a protein composition-based approach in evaluating (plant) protein digestibility.

The diamondback moth, Plutella xylostella, is a major pest of brassica vegetables and oilseed crops, posing a serious threat to China’s grain and oil production. RNA interference (RNAi) has been developed as an efficient strategy to control pests. In this study, the effects of RNAi on P. xylostella were evaluated by injecting two doses of synthesized dsPxvATPasea. The transcripts of PxvATPasea were widely transcribed during different developmental stages from egg to adult. They were abundantly expressed in the hindgut and Malpighian tubules, compared with other tissue types. Introduction of 800 ng dsPxvATPasea in the fourth-instar larvae greatly reduced corresponding mRNA levels by 3.1 and 1.4 times on day 2 and 3, respectively, causing 66.6% mortality and 33.4% treated larvae pupated. Silencing PxvATPasea by injecting 1200 ng dsRNA significantly decreased the expression level by 5.0 and 2.0 times on the second and third day, leading to 79.2% larval lethality and 20.8% depleted larvae pupated. Moreover, introducing 800 ng or 1200 ng dsPxvATPasea finally reduced larval fresh weight by 22.1% and 28.8%, respectively. The results indicated that the silencing efficiency of PxvATPasea worked in a dose-dependent way. Consequently, PxvATPasea is a potential molecular target gene. Our findings will facilitate the application of RNAi technology to manage P. xylostella.

Schizochytrium sp. is a marine microorganism used for the commercial production of docosahexaenoic acid (DHA), with applications in the food and feed industries. In addition to DHA, Schizochytrium sp. has the ability to produce trace amounts of carotenoids, including beta-carotene and astaxanthin. This study aims to enhance astaxanthin production in Schizochytrium sp. through optimization of culturing conditions. Contrary to previous reports, supplementation with ethanol or isopropanol did not obviously affect astaxanthin production. Notably, reducing nitrogen levels and replacing glucose with fructose led to a 24-fold increase in astaxanthin production (139.3 mu g/g dry cell weight) compared to the lipid fermentation conditions. Transcriptomic analysis indicated that upregulation of genes involved in the mevalonate pathway, including acat, crtE, and crtIBY, alongside the beta-carotene hydroxylase gene crtZ, probably contributed to the enhanced astaxanthin production. These findings offer a straightforward and efficient approach for improving astaxanthin production in Schizochytrium sp., thereby enhancing its potential applications in the food and feed industries.

This work aims to verify the feasibility of improving protein function by regulating its hydrophobicity and reveal the relationship between structure and function. Whey protein (WP) and zein were the source of hydrophilic and hydrophobic polypeptide chains to prepare complex proteins (CPs) with much different structure and function. The results showed that the water- and oil-holding capacities, emulsifying properties and gel properties of CPs can be significantly improved via changing WP-zein ratio. All these can be attributed to the changes in protein hydrophobicity, which not only regulated the binding strength of protein to water and oil, but also modified their molecular structure (surface characteristics, availability of free thiols, α-helix, β-sheet, random coil and the formation of disulfide bonds). Notably, optimal protein hydrophobicity varies greatly among different functional properties. Overall, the techno-functional properties of protein can be improved via tuning its hydrophobicity, which may provide novel sights in protein modification.

pH-driven method is an effective strategy to prepare complex protein. This study provides guidance on how to select acidifiers and alkalizers from view of Hofmeister ion effects. Cations and anions regulated the molecular structure (particle size, surface charge, protein folding/unfolding, structural orderliness) of complex rapeseed proteins (CRPs) mainly via electrostatic and hydrogen bond. No evident changes were found in the molecular weight distribution, but their distribution on oil/air-water interface varied greatly. Various techno-functional properties of CRPs were synergistically improved: Citrate3- and Na+ increased the emulsifying activity index of CRPs from 80 to 102.21 m2/g; Citrate3-, K+ and Na+ made the foaming stability of CRPs close to 80 % after 60 min of storage. Moreover, the oil/water-holding and gel properties of CRPs were regulated effectively. These findings demonstrate the key role of Hofmeister ion effects in improving CRPs properties, contributing to develop, select, and apply novel acidifiers and alkalizers during pH-driven treatment.

Regulation in protein-polyphenols interaction contributes to their application in emulsion, but relative information is very limited. Therefore, this work designed a novel strategy to regulate the interaction of rapeseed protein (RP) with quercetin (Que) for stabilizing high internal phase emulsions (HIPEs) loaded with vitamin D3 (VD3). The results showed that whey protein enhanced the interaction of RP with water molecular, and zein contributed to its binding with Que. Modified RP-Que complex possessed higher Que-binding rate (76.15 % vs 96.86 %), DPPH· scavenging rate (45.33 % vs 74.89 %), surface charge (-24 mV), orderliness as well as smaller size (200 nm). Above improvements increased its diffusion rate (0.74 mN/m/s1/2), modulus and adsorption content (350 %) on the oil-water interface, which formed denser network structure in HIPEs, and gave them higher bioavailability and stability of VD3 and storage stability of 3D-printing products. Overall, molecular regulation improved the application potential of RP-Que complex in HIPEs.

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Our previous experiments found that rapeseed protein (RP) has applicability in low-moisture textured proteins. The amount of RP added is limited to <20 %, but the addition of 20 % RP still brings some negative effects. Therefore, in order to improve the quality of 20%RP textured protein, this experiment added different proportions of sodium tripolyphosphate (STPP) to improve the quality of the product, and studied the physicalchemical properties and molecular structure changes of the product to explore the possible modification mechanism. The STPP not only improved the expansion characteristics of extrudates, but also increased the brightness of the extrudates, the rehydration rate. In addition, STPP increased the specific mechanical energy during extrusion, decreased the material mass flow rate. Furthermore, STPP decreased the starch digestibility, increased the content of slow-digesting starch and resistant starch. STPP increased the degree of denaturation of extrudate proteins, the proportion of beta -sheets in the secondary structure of proteins, as well as the intermolecular hydrogen bonding interactions. The gelatinization degradation degree of starch molecules also decreased with the addition of STPP. STPP also increased the protein-starch interactions and enhanced the thermal stability of the extrudate. All these indicate that STPP can improve the physical-chemical properties of extrudate.

Astaxanthin esters are a major form of astaxanthin found in nature. However, the exact mechanisms of the biosynthesis and storage of astaxanthin esters were previously unknown. We found that Schizochytrium sp. synthesized both astaxanthin and docosahexaenoic acid (DHA)-enriched lipids. The major type of astaxanthin produced was free astaxanthin along with astaxanthin-DHA monoester and other esterified forms. DHA accounted for 41.0% of the total fatty acids from astaxanthin monoesters. These compounds were deposited mainly in lipid droplets. The biosynthesis of the astaxanthin esters was mainly carried out by a novel diacylglycerol acyltransferase ScDGAT2-1, while ScDGAT2-2 was involved only in the production of triacylglycerol. We also identified astaxanthin ester synthases from the astaxanthin-producing algae Haematococcus pluvialis and Chromochloris zofingiensis, as well as a thraustochytrid Hondaea fermentalgiana with an unknown carotenoid profile. This investigation enlightens the application of thraustochytrids for the production of both DHA and astaxanthin and provides enzyme resources for the biosynthesis of astaxanthin esters in the engineered microbes.

Low bioavailability of quercetin (Que) reduces its preclinical and clinical benefits. In order to improve Que bioavailability, a novel whey protein isolate (WPI)-zein nanogel was prepared by pH -driven self -assembly and heat -induced gelatinization. The results showed that hydrochloric acid can be substituted by both acetic acid and citric acid during the pH -driven process. After encapsulation, the bioavailability of Que in nanogels (composed of 70 % WPI) induced by different acidifiers increased to 19.89 % (citric acid), 21.65 % (hydrochloric acid) and 24.34 % (acetic acid), respectively. Comparatively, nanogels induced by acetic acid showed higher stability (pH and storage stability), re-dispersibility (75.62 %), Que bioavailability (24.34 %), and antioxidant capacity (36.78 % for DPPH scavenging rates). s improved performance of nanogels. In mechanism, acetic acid significantly balanced different intermolecular forces by weakening " acid -induced denaturation " effect. Moreover, the faster binding of Que and protein as well as higher protein molecular flexibility and randomness (higher ratio of random coil) was also observed in nanogels induced by acetic acid. All of these changes contributed to improve nanogels performances. Overall, WPI-zein nanogels induced by acetic acid might be a safe, efficiency and stable delivery system to improve the bioavailability of hydrophobic active ingredients.

The market for functional and nutraceutical foods is one of the fastest growing markets in the new food creation area, especially in the era of great health. Because of its nutritional and functional qualities, plant-based fermented milk has great development prospects. This review summarizes the quality characteristics of plant-based raw materials, probiotic fermentation characteristics, and the effects of probiotics on the flavor, nutrition and gel stability of plant-based fermented milk during the fermentation process. Additionally, we discuss future prospects for the development and application of plant-based fermented milk. This review aims to offer scientific guidelines for the production of high-quality plant-based fermented milk.

To improve the techno-functional properties of rapeseed protein (RP), this work tried to regulate the molecular structure of RP via inducing the co-assembly of RP with zein and whey protein (WP). The results showed that WP and zein mainly regulate the folding process of RP through hydrophobic and disulfide bonds, thereby altering the structural conformation and forming stable complex RP (CRP). WP addition not only increased the number of surface charges and hydrophilicity of proteins, but also decreased their sizes, improved the water solubility, as well as the availability of active groups. These changes significantly increased the foaming capacity (from 60 % to 147 %) and in vitro gastric digestion rate (from 10 % to 60 %) of CRP. Besides, WP also contributed to the formation of gels and the regulation of their textural profiles. Comparatively, zein improved the hydrophobicity of CRP and balanced degree of intermolecular forces, which effectively increased the emulsifying activity index of CRP from 22 m2/g to 90 m2/g. Zein decreased the hardness, springiness and water-holding capacity of gel, but increased its gumminess and chewiness. Overall, both WP and zein effectively changed the structural conformation of RP, and improved its techno-functional properties, which provides an effective strategy to modify protein.

N-3 long-chain polyunsaturated fatty acids (n-3 LCPUFAs) are essential for physiological requirements and disease prevention throughout life but are not adequately consumed worldwide. Dietary supplementation with plant-derived α-linolenic acid (ALA) has the potential to rebalance the fatty acid profile and enhance health benefits but faces challenges such as high β-oxidation consumption, low hepatic conversion efficiency, and high oxidative susceptibility under stress. This review focuses on the metabolic fate and potential regulatory targets of ALA-containing lipids in vivo, specifically the pathway from the gastrointestinal tract to the lymph, blood circulation, and liver. We propose a hypothesis that positively regulates the conversion of ALA into n-3 LCPUFAs based on the model of "fast" or "slow" absorption, transport, and hepatic metabolic fate. Furthermore, the potential effects of dietary nutrients on the metabolic conversion of ALA into n-3 LCPUFAs are discussed. The conversion of ALA is differentially regulated by structured lipids, phospholipids, other lipids, carbohydrates, specific proteins, amino acids, polyphenols, vitamins, and minerals. Future research should focus on designing a steady-state and precise delivery system for ALA, coupled with specific nutrients or phytochemicals, to effectively improve its metabolic conversion and ultimately achieve synergistic regulation of nutrition and health effects.

This study investigated the potential of rapeseed protein as a novel alternative protein source in meat analogs produced by extrusion cooking technology. To study the substitutability of rapeseed protein to soybean protein, the addition ratios of rapeseed protein to soybean protein in the extrusion experiment were 0:50, 10:40, 20:30, 30:20, 40:10 and 50:0 (w/w), respectively. The results showed that adding 0–20% rapeseed protein not only decreased the specific mechanical energy and mass flow rate of the mixture during extrusion, but also enhanced the hardness and chewiness of the extrudates while reducing their elasticity and resilience. In addition, increasing the amount of rapeseed protein may lead to decreased expansion characteristics, internal pore structure, and water absorption rate of the extrudates, as well as a decrease in brightness and an increase in redness on the surface. Weibull model could well describe the rehydration behavior for dynamic fitting of the extrusion rehydration process. Moreover, with the increase of rapeseed protein substitution, the content of disulfide bonds, and the proportion of relatively ordered protein secondary structures with high stability increased, and the degree of protein denaturation decreased. Besides, the degree of starch gelatinization reduced and the thermal stability of extrudates improved. Interaction analysis indicated that disulfide bonds are the primary force maintaining protein-mediated interactions in the extrudates. The incorporation of rapeseed protein shortened the mean residence time of molten feedstock and shifted the flow state to plug flow. Overall, our investigation suggests that within low levels of addition (≤20%, w/w), rapeseed protein has the potential to partially substitute for the soybean protein-based textured proteins. High substitution levels or complete substitution are not feasible due to the relatively low expansion and rehydration rates.

Astaxanthin is a highly value-added keto-carotenoid compound. The astaxanthin 3S,3'S-isomer is more desirable for food additives, cosmetics, and pharmaceuticals due to health concerns about chemically synthesized counterparts with a mixture of three isomers. Biosynthesis of 3S,3'S-astaxanthin suffers from limited content and productivity. We engineered Yarrowia lipolytica to produce high levels of 3S,3'S-astaxanthin. We first assessed various β-carotene ketolases (CrtW) and β-carotene hydroxylases (CrtZ) from two algae and a plant. HpCrtW and HpCrtZ from Haematococcus pluvialis exhibited the strongest activity in converting β-carotene into astaxanthin in Y. lipolytica. We then fine-tuned the HpCrtW and HpCrtZ transcriptional expression by increasing the rounds of gene integration into the genome and applied a modular enzyme assembly of HpCrtW and HpCrtZ simultaneously. Next, we rescued leucine biosynthesis in the engineered Y. lipolytica, leading to a five-fold increase in biomass. The astaxanthin production achieved from these strategies was 3.3 g/L or 41.3 mg/g dry cell weight under fed-batch conditions, which is the highest level reported in microbial chassis to date. This study provides the potential for industrial production of 3S,3'S-astaxanthin, and this strategy empowers us to build a sustainable biorefinery platform for generating other value-added carotenoids in the future.

Schizochytrium sp. is commercially used for the production of docosahexaenoic acid (DHA). Some strains of Schizochytrium sp. are also known to produce low amounts of carotenoids, including astaxanthin and β-carotene. In order to enhance the production of astaxanthin in Schizochytrium sp., we established a seamless genome editing system with a dual selection marker for rapid screening of positive transformants. By using this system, we strengthened the endogenous mevalonate pathway, enhanced the supply of geranylgeranyl diphosphate and β-carotene, upregulated endogenous β-carotene hydroxylase, and introduced the algal astaxanthin pathway. The highest astaxanthin production in the engineered Schizochytrium sp. was achieved at 8.1 mg/L (307.1 μg/g dry cell weight) under shake-flask conditions, which was 2.6-fold higher than that in the start strain. Meanwhile, the percentage of DHA to total fatty acids was not obviously affected. We then eliminated the dual selection marker by using the Cre-loxP recombination system, and the engineered strain was ready for iterative editing. The developed system could be applied to seamlessly engineer DHA-producing Schizochytrium sp. toward astaxanthin and other value-added terpenoids, which broadens the application of this strain.