Tailored UPRE2 variants for dynamic gene regulation in yeast.

Genetic elements are foundational in synthetic biology serving as vital building blocks. They enable programming host cells for efficient production of valuable chemicals and recombinant proteins. The unfolded protein response (UPR) is a stress pathway in which the transcription factor Hac1 interacts with the upstream unfolded protein response element (UPRE) of the promoter to restore endoplasmic reticulum (ER) homeostasis. Here, we created a UPRE2 mutant (UPRE2m) library. Several rounds of screening identified many elements with enhanced responsiveness and a wider dynamic range. The most active element m84 displayed a response activity 3.72 times higher than the native UPRE2. These potent elements are versatile and compatible with various promoters. Overexpression of HAC1 enhanced stress signal transduction, expanding the signal output range of UPRE2m. Through molecular modeling and site-directed mutagenesis, we pinpointed the DNA-binding residue Lys60 in Hac1(Hac1-K60). We also confirmed that UPRE2m exhibited a higher binding affinity to Hac1. This shed light on the mechanism underlying the Hac1-UPRE2m interaction. Importantly, applying UPRE2m for target gene regulation effectively increased both recombinant protein production and natural product synthesis. These genetic elements provide valuable tools for dynamically regulating gene expression in yeast cell factories.

Anti-diabetic mechanism and potential bioactive peptides of casein hydrolysates in STZ/HFD-induced diabetic rats.

BACKGROUND: Casein hydrolysates have attracted much interest as anti-diabetic food, but their hypoglycemic mechanism and biopeptides are not well understood. This study aimed to explore the anti-diabetic mechanism and potential biopeptides of casein hydrolysates in streptozotocin/high-fat-diet-induced diabetic rats and HepG2 cells. RESULTS: Oral administration of casein hydrolysate prepared with papain-Flavourzyme combination (P-FCH) decreased fasting blood glucose, improved oral glucose tolerance, and reduced HbA1c values in diabetic rats. P-FCH was ineffective in alleviating insulin resistance (homeostasis model assessment and insulin sensitivity index) and enhancing hepatic insulin signaling transduction (phosphorylated Akt, hexokinase activity, and pyruvate kinase activity) in diabetic rats. However, P-FCH significantly upregulated adenosine monophosphate-activated protein kinase phosphorylation and glucose transporter-2 expression, inhibited phosphoenolpyruvate carboxylase kinase activity, and elevated glycogen content in liver tissue of diabetic rats. Furthermore, P-FCH increased glucose consumption independently in normal and insulin-resistant HepG2 cells without the presence of insulin. The peptide composition of P-FCH was characterized. The potential biopeptides in P-FCH showed the sequence characteristic of a Val at the N-terminal or a Pro at the P2 position, and the hypoglycemic activity of Val-Pro-Leu-Gly (the most potential biopeptide in P-FCH) was verified by oral glucose tolerance test in mice. CONCLUSION: These results suggested that activation of the non-insulin-mediated AMPK pathway might be the determinant mechanism of P-FCH on the hypoglycemic effect. The novel peptide Val-Pro-Leu-Gly in P-FCH was effective in reducing blood glucose levels when orally administered to mice. © 2023 Society of Chemical Industry.

Effects of physical method and enzymatic hydrolysis on the properties of soybean fiber-rich stabilizer for oil in water emulsions.

BACKGROUND: Okara is a by-product from the soybean industry and an abundant resource of insoluble soybean fiber (ISF). ISF with various properties could be obtained by different extraction methods. It is an attractive option to utilize okara by taking advantage of ISF as an emulsifier or stabilizer. RESULTS: Compared with the untreated ISF (ISFUT ), superfine grinding reduced the particle size and viscosity of ISF (ISFSG ). Steam explosion increased the water solubility from 17.5% to 51.7% but decreased the water holding capacity and swelling capacity of ISF (ISFSE ) from 15.0 and 14.0 g/g to 4.2 and 3.3 g/g, respectively. Emulsions prepared by ISFUT and ISFSG before or after enzymatic hydrolysis presented large oil droplets and were unstable. Although emulsions prepared by ISFSE after enzymatic hydrolysis (ISFSE-E ) showed flocculation, the volume-weighted average diameter (19.7 µm) were the smallest while the viscosity and viscoelastic modulus were the highest, and exhibited excellent physical stability during storage. CONCLUSION: ISF obtained by physical and hydrolysis treatment displayed diverging physicochemical properties while ISF prepared by steam explosion-enzymatic hydrolysis presented the best potential to stabilize emulsions. The present study could provide novel information about the utilization of okara by the application of ISF as an emulsifier or stabilizer. © 2023 Society of Chemical Industry.

Advances in recombinant protease production: current state and perspectives.

Proteases, enzymes that catalyze the hydrolysis of peptide bonds in proteins, are important in the food industry, biotechnology, and medical fields. With increasing demand for proteases, there is a growing emphasis on enhancing their expression and production through microbial systems. However, proteases' native hosts often fall short in high-level expression and compatibility with downstream applications. As a result, the recombinant production of proteases has become a significant focus, offering a solution to these challenges. This review presents an overview of the current state of protease production in prokaryotic and eukaryotic expression systems, highlighting key findings and trends. In prokaryotic systems, the Bacillus spp. is the predominant host for proteinase expression. Yeasts are commonly used in eukaryotic systems. Recent advancements in protease engineering over the past five years, including rational design and directed evolution, are also highlighted. By exploring the progress in both expression systems and engineering techniques, this review provides a detailed understanding of the current landscape of recombinant protease research and its prospects for future advancements.

Improved protein production in yeast using cell engineering with genes related to a key factor in the unfolded protein response.

The yeast Saccharomyces cerevisiae is a widely used cell factory for protein production. Increasing the protein production capacity of a yeast strain may be beneficial for obtaining recombinant proteins as a product or exerting its competence in consolidated bioprocessing. However, heterologous protein expression usually imposes stress on cells. Improving the cell's ability to cope with stress enhances protein yield. HAC1 is a key transcription factor in the unfolded protein response (UPR). In this study, several genes related to the UPR signal pathway, including unfolded protein sensing, HAC1 mRNA splicing, mRNA ligation, mRNA decay, translation, and Hac1p degradation, were selected as targets to engineer yeast strains. The final engineered strain produced α-amylase 3.3-fold, and human serum albumin 15.3-fold, greater than that of the control strain. Key regulation and metabolic network changes in the engineered strains were identified by transcriptome analysis and physiological characterizations. This study demonstrated that cell engineering with genes relevant to the key node HAC1 in UPR increased protein secretion substantially. The verified genetic modifications of this study provide useful targets in the construction of yeast cell factories for efficient protein production.

Six categories of amino acid derivatives with potential taste contributions: a review of studies on soy sauce.

During the fermentation of soy sauce, the metabolism of microorganisms and the Maillard reaction produce a wide variety of metabolites that contribute to the unique and rich flavor characteristics of soy sauce, such as amino acids, organic acids and peptides. Amino acid derivatives, a relatively new taste compounds, formed by the reaction of enzymes or non-enzymes from sugars, amino acids, and organic acids released through metabolism by microorganisms during soy sauce fermentation, have begun to gain more and more attention in recent years. This review focused on our existing knowledge of the sources, taste characteristics and synthesis methods of the 6 categories of amino acid derivatives, including Amadori compounds, γ-glutamyl peptides, pyroglutamyl amino acids, N-lactoyl amino acids, N-acetyl amino acids and N-succinyl amino acids. Sixty-four amino acid derivatives were detected in soy sauce, of which 47 were confirmed to have potential contribution to the taste of soy sauce, especially umami and kokumi, and some of them also have the effect of reducing bitterness. Furthermore, some amino acid derivatives, like γ-glutamyl peptides and N-lactoyl amino acids, were found to be synthesized enzymatically in vitro, which laid the foundation for further study on their formation pathways in the future.

Preparation and characterization of emulsion gels stabilized by adequately preprocessed insoluble soybean fiber from okara.

Emulsion gels as soft materials were formulated by insoluble soybean fiber (ISF) assembled from okara in this study. Steam explosion on okara (ISFS) transformed the insoluble fiber in the original okara (ISFU) into soluble fiber. Enzymatic hydrolysis led to a lower protein content, smaller particle size and smaller contact angle of ISF. ISFE, which was obtained by enzymatic hydrolysis of ISFU, was not able to produce stable emulsion gels at 0.50 to 1.50 wt% ISF, whereas the ISF after a combined steam explosion-enzymatic hydrolysis treatment (giving rise to ISFSE) stabilized emulsion gels at varying oil volume fractions (φ) from 10 to 50%. The ζ-potential of emulsion gels was around -19 to -26 mV. The droplet size first decreased (from 43.8 µm to 14.8 µm when at φ = 0.3) with increasing ISF content (from 0.25 wt% to 1.25 wt%) and then remained constant, as also seen from the microstructure. The apparent viscosity and viscoelastic properties were strengthened upon increasing both the ISF concentration and oil volume fraction. The protein and soluble fiber contributed to the interfacial activity of ISF while the insoluble fiber played an important role in the gel-like structured network of emulsion gels, making them maintain physical stability during long term storage. These findings could provide novel information about soybean fiber to fabricate soft materials and the utilization of okara at an industrial-scale.

Exploring the Release of Elastin Peptides Generated from Enzymatic Hydrolysis of Bovine Elastin via Peptide Mapping.

To enhance the understanding of enzymatic hydrolysis and to accelerate the discovery of key bioactive peptides within enzymatic products, this research focused on elastin as the substrate and investigated the variations in peptide profiles and the production of key bioactive peptides (those exceeding 5% of the total) and their impacts on the biological activity of the hydrolysates. Through the application of advanced analytical techniques, such as stop-flow two-dimensional liquid chromatography and ultra-high-performance liquid chromatography-tandem mass spectrometry, the research tracks the release and profiles of peptides within elastin hydrolysates (EHs). Despite uniform peptide compositions, significant disparities in peptide concentrations were detected across the hydrolysates, hinting at varying levels of bioactive efficacy. A comprehensive identification process pinpointed 403 peptides within the EHs, with 18 peptides surpassing 5% in theoretical maximum content, signaling their crucial role in the hydrolysate's bioactivity. Of particular interest, certain peptides containing sequences of alanine, valine, and glycine were released in higher quantities, suggesting Alcalase® 2.4L's preference for these residues. The analysis not only confirms the peptides' dose-responsive elastase inhibitory potential but also underscores the nuanced interplay between peptide content, biological function, and their collective synergy. The study sets the stage for future research aimed at refining enzymatic treatments to fully exploit the bioactive properties of elastin.

Flavor differences of soybean and defatted soybean fermented soy sauce and its correlation with the enzyme profiles of the kojis.

BACKGROUND: Soybeans and defatted soybeans, commonly used as protein ingredients, have different flavors of their fermented soy sauce. Clarifying the differences between the two soy sauces, as well as the formation mechanism, is an important prerequisite for improving the flavor of defatted soybean soy sauce. To this goal, the aroma characteristics of two soy sauces and their volatile profiles were compared by sensory evaluation and gas chromatography-mass spectrometry, and eight enzyme activities and volatile profiles of matured koji were determined. RESULTS: Sensory results showed that the acids, fruity and cooked potato-like attributes were higher in whole soybean fermented soy sauce, whereas defatted soybean soy sauce exhibited higher smoky and malty attributes, closely related to the contents of aroma-active compounds in soy sauce, such as isobutyl acetate, 2/3-methylbutanal, acetic acid and 2/3-methylbutanoic acid. The content of most volatiles in the matured kojis showed a consistent trend with that of soy sauce: alcohols, acids, furan(one)s and ketones. Interestingly, acid protease and cellulase activities were 3.3 and 1.6 times higher in the whole soybean koji than in defatted soybean koji, respectively, whereas neutral protease, aminopeptidase, glucoamylase and ß-glucosidase were approximately 2.0 times higher in defatted soybean koji. CONCLUSION: In summary, the flavor differences between soybean and defatted soybean fermented soy sauce were not only caused by the differences in the content of flavor precursors in the materials, but also closely related to the differences in the enzymatic profiles accumulated during the koji-making process. © 2022 Society of Chemical Industry.

Hydration effects of insoluble soybean fiber (ISF) on rheological properties and freeze-thaw stability of ISF concentrated emulsions.

BACKGROUND: Concentrated emulsions have been formulated in many foods. The insoluble soybean fiber (ISF) can be utilized as a particle to stabilize concentrated emulsions. However, the approach to control the rheological properties and stability of the ISF concentrated emulsions is still worth investigating. RESULTS: In this study, alkali-extracted ISF was hydrated by adding sodium chloride or heating and the prepared concentrated emulsions were subjected to freeze-thawing. Compared with the original hydration method, salinization reduced the absolute ζ-potential of the ISF dispersions to 6 mV, resulting in a lower absolute ζ-potential of the concentrated emulsions, which led to a decreased electrostatic repulsion and the largest droplet size, but to the lowest apparent viscosity, viscoelastic modulus, and stability. By contrast, hydration by heating promoted the interparticle interactions, and then a decreased droplet size (54.5 µm) but with a more densely distributed droplets was observed, together with enhanced viscosity and viscoelasticity properties. The fortified network structure improved the stability of the concentrated emulsions both against high-speed centrifugation and long-term storage. Additionally, secondary emulsification after freeze-thaw further improved the performance of the concentrated emulsions. CONCLUSION: The results suggest that the formation and stability of the concentrated emulsion could be regulated by different hydration methods of particles, which could be adjusted according to the practical applications. © 2023 Society of Chemical Industry.

A systematic review on the flavor of soy-based fermented foods: Core fermentation microbiome, multisensory flavor substances, key enzymes, and metabolic pathways.

The characteristic flavor of fermented foods has an important impact on the purchasing decisions of consumers, and its production mechanisms are a concern for scientists worldwide. The perception of food flavor is a complex process involving olfaction, taste, vision, and oral touch, with various senses contributing to specific properties of the flavor. Soy-based fermented products are popular because of their unique flavors, especially in Asian countries, where they occupy an important place in the dietary structure. Microorganisms, known as the souls of fermented foods, can influence the sensory properties of soy-based fermented foods through various metabolic pathways, and are closely related to the formation of multisensory properties. Therefore, this review systematically summarizes the core microbiome and its interactions that play an active role in representative soy-based fermented foods, such as fermented soymilk, soy sauce, soybean paste, sufu, and douchi. The mechanism of action of the core microbial community on multisensory flavor quality is revealed here. Revealing the fermentation core microbiome and related enzymes provides important guidance for the development of flavor-enhancement strategies and related genetically engineered bacteria.

Current trends in the anti-photoaging activities and mechanisms of dietary non-starch polysaccharides from natural resources.

Photoaging is a complex and multistage process triggered mainly by ultraviolet (UV) radiation due to exposure to sunlight. Photoaging induces DNA damage and oxidative stress that initiate an inflammatory response and an increase of matrix metalloproteinases (MMPs) expression, which results in cumulative changes in skin appearance, structure, and functions, and eventually causes skin carcinogenesis. Dietary polysaccharides from bio-resources have been utilized as functional ingredients in healthy food, cosmetics, and drug due to their good bioactivities. However, a systematic introduction to their effects and underlying mechanisms in anti-photoaging is limited. This review discusses the damage and pathogenesis of UV-induced photoaging and summarizes the up-to-date advances in research on the anti-photoaging activity of non-starch polysaccharides from natural edible resources considering the influence of oxidative stress, DNA damage, MMPs regulation, inflammation, and melanogenesis, primarily focusing on the cellular and molecular mechanisms. This paper will help to understand the anti-photoaging functions of dietary non-starch polysaccharides from natural resources and further application in drug and functional food.

Theragra chalcogramma Hydrolysates, Rich in Gly-Leu-Pro-Ser-Tyr-Thr, Exerts Anti-Photoaging Potential via Targeting MAPK and NF-κB Pathways in SD Rats.

Previous studies have revealed that excessive exposure to UV irradiation is the main cause of skin photoaging and the signaling pathways of MAPK and NF-κB are involved in this progression. The present study aims to investigate the anti-photoaging effects of low molecular weight hydrolysates from Theragra chalcogramma (TCH) and to clarify the underlying mechanism. The degradation of mechanical barrier functions in photoaged skin was substantially ameliorated after TCH administration; meanwhile, TCH significantly elevated the antioxidant capacity and suppressed the over-production of inflammatory cytokine IL-1ß. Moreover, the histopathological deteriorations such as epidermal hyperplasia and dermal loss were significantly alleviated, along with the increase in procollagen type I content and decrease in MMP-1 activity (p < 0.05). Furthermore, TCH effectively blocked the MAPK and NF-κB signaling pathways through inhibition of the phosphorylation of p38, JNK, ERK, iκB, and p65 proteins. Collectively, these data indicate that TCH has potential as a novel ingredient for the development of anti-photoaging foods.

Theragra chalcogramma Hydrolysate, Rich in Gly-Leu-Pro-Ser-Tyr-Thr, Alleviates Photoaging via Modulating Deposition of Collagen Fibers and Restoration of Extracellular Components Matrix in SD Rats.

Excessive exposure of the skin to ultraviolet irradiation induces skin photoaging, which seriously deteriorates the barrier functions of skin tissue, and even causes skin damages and diseases. Recently, dietary supplements from marine sources have been found to be useful in modulating skin functions and can be used to alleviate photoaging. Herein, the low-molecular-weight hydrolysates with a photoaging-protection effect were prepared by enzymatic hydrolysis from Theragra chalcogramma (TCH), and the potential mechanism were subsequently explored. The results revealed that TCH desirably improved the barrier functions of photoaged skin and stimulated the deposition of ECM components Col I, Hyp, and HA in the dermal layer. Histologically, TCH reduced the epidermal hyperplasia and restored the impaired architectures in a dose-dependent manner. Meanwhile, the activity of matrix metalloproteinase-1 (MMP-1) in photoaging skin was inhibited, and the expression levels of elastin and fibrillin-1 were elevated accordingly after TCH administration, and the significant improvements were observed at high-dose level (p < 0.05). Taken together, the efficacy of TCH against skin photoaging is highly associated with the regulation on ECM metabolism and the repairing of damaged mechanical structure.

Tripeptide Leu-Ser-Trp Regulates the Vascular Endothelial Cells Phenotype Switching by Mediating the Vascular Smooth Muscle Cells-Derived Small Extracellular Vesicles Packaging of miR-145.

Tripeptide LSW, initially identified as a potent ACE inhibitory peptide from soybean protein, was recently reported to exert a protective effect against angiotensin II-induced endothelial dysfunction via extracellular vesicles (EVs). However, the molecular mechanisms, especially in lipid accumulation-induced atherosclerosis, still remain unclear. The study aimed to investigate whether the protective effects of LSW against endothelial dysfunction on vascular endothelial cells (VECs) was via vascular smooth muscle cells (VSMCs)-derived miRNA-145 packaged in EVs. The miRNA-145 was concentrated in EVs from LSW-treated VSMCs (LEVs), internalized into the HVUECs, and targeted the programmed cell death protein 4 (PDCD4) expression of HUVECs. Oxidized low-density lipoprotein (oxLDL) was applied to induce endothelial dysfunction in HUVECs; oxLDL-induced endothelial dysfunction in HUVECs was attenuated by PDCD4 knockout or LEVs incubation. The results of this study suggested a novel function of LSW as a regulator on the functional EVs from vascular cells in the oxLDL-induced atherosclerotic model.

Construction of in vitro fermentation model using gut microbiota relating to glucose and lipid metabolism: a supplementary method for initial screening of polysaccharides with hypoglycemic potentials.

BACKGROUND: Besides in vitro fecal fermentation model, a few supplementary methods have been constructed for high-throughput screening of polysaccharides with hypoglycemic potentials. The purpose of this study was to establish a co-culture fermentation model constructed by gut microbiota relating to glucose and lipid metabolism as a supplementary method for comparatively evaluating the proliferative effects and hypoglycemic potentials of typical plant polysaccharides, e.g. konjac glucomannan, Lycium barbarum L. polysaccharide, oat glucan and alga-derived fucoidan. RESULTS: The results showed that the mixing culture medium of butyrate-producing bacteria, Bacteroides, Bifidobacterium and Lactobacillus at a ratio of 50:40:9:1 was optimal. This testing model in line with quantitative polymerase chain reaction (qPCR) and metabolite analysis multi-dimensionally differentiated four polysaccharides possessing different behaviors on proliferation of total bacteria and specific genus or strain and accumulation of short chain fatty acids. CONCLUSION: Our study provided crucial data for establishing an initial screening method for proliferative effect/specific structure-oriented extraction of polysaccharide with hypoglycemic potential. © 2022 Society of Chemical Industry.

Emulsifying and whipping properties of mixing polysaccharide dispersions: effect of ratio between insoluble soybean fiber and hydroxypropyl methylcellulose.

BACKGROUND: The interactions between various food colloids in different systems (e.g., dispersions, emulsions, creams) have a bearing on the processing and characteristics of food systems. Hydrophilic polysaccharides have been proven to have the potential to fabricate the above systems. In the present work, hydroxypropyl methylcellulose (HPMC) was partially replaced by the insoluble soybean fiber (ISF) extracted from defatted okara to prepare mixing dispersions, oil-in-water emulsions and whipped creams. RESULTS: The presented work showed that as the proportion of ISF increased, the foaming properties of ISF/HPMC dispersions were enhanced, the absolute value of the ζ-potential and the particle size of the emulsions increased, while the heat stability and centrifugal stability first increased and then decreased. Upon whipping, the loss angle (tan δ) decreased first and then increased, while the overrun, foam stability and cream stability, as well as the elastic modulus (G'), presented the opposite trend. CONCLUSION: These results indicated that an appropriate amount (40-60%) of ISF in the ISF/HPMC systems enhanced the foaming and emulsifying capacities of mixtures and the stability of the resultant emulsion; subsequently, the whipping performance and whipped cream network structure were strengthened, suggesting that ISF has great potential for application in whipped cream as a 'green' and safe food ingredient. © 2022 Society of Chemical Industry.

Carboxymethyl cellulose/okara protein influencing microstructure, rheological properties and stability of O/W emulsions.

BACKGROUND: The role of protein-polysaccharide interactions and their mixtures has been a vital factor affecting the formation and stability of food emulsions. Okara protein (OP), which is extracted from the by-product of soybean processing, has received much attention because of its abundant sources and potential attributes with respect to food formulation. Carboxymethyl cellulose (CMC), a well-known food-grade polysaccharide additive, has been widely utilized in the protein-polysaccharide system, whereas, among the proteins, the role of OP has not yet been explored. RESULTS: The present study first assessed the ζ-potential and hydrodynamic diameter of aqueous mixtures containing OP (1.0 wt%) and CMC (0-0.5 wt%), followed by the investigation of OP-CMC mixtures stabilized O/W emulsions. As CMC increased, oil droplet size, surface protein adsorption, apparent viscosity and storage modulus increased, whereas the loss tangent decreased. CONCLUSION: CMC resulted in emulsion destabilization compared to emulsions without CMC, whereas a higher concentration of CMC promoted emulsion stability against creaming for emulsions in the presence of CMC. The results provide information with respect to OP and CMC being incorporated into food formulations and also strengthen our understanding of the related mechanism, in addition to facilitating the further utilization of OP. © 2020 Society of Chemical Industry.

Physicochemical Characterization of Hizikia fusiforme Polysaccharide and Its Hypoglycemic Activity via Mediating Insulin-Stimulated Blood Glucose Utilization of Skeletal Muscle in Type 2 Diabetic Rats.

In the current study, a functional polysaccharide fraction (HFP) was obtained from Hizikia fusiforme by ultrasound-assisted enzymatic extraction, and its structural characterization and hypoglycemic activity and potential molecular mechanism were investigated. The results indicated that HFP with high uronic acid was a heterogeneous polysaccharide composed of six monosaccharides. Congo red test explained that HFP had no triple helix conformation. AFM analysis revealed that HFP was spherical particle with flame-like aggregates and multiple strands closely arranged. Rheological analysis showed that HFP exhibited shear-thinning flow behavior. HFP significantly ameliorated diabetes-related symptoms and serum profiles and increased muscle glycogen storage in rats. HFP administration at 400 mg/kg body weight/day displayed greater advantages than metformin in controlling the levels of fasting blood glucose, triglyceride (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bile acid (TBA) of diabetic rats. Intervention of HFP up-regulated markedly the expression of AMPK-α, GLUT4, PI3K and Akt in skeletal muscle of diabetic rats at the mRNA and protein levels, revealing hypoglycemic effects of HFP may be related closely to improving insulin resistance and mitochondrial function of skeletal muscle.

Identification and function of penaeidin 3 and penaeidin 5 in Fenneropenaeus merguiensis.

Antimicrobial peptides (AMPs) participate in immune defenses of invertebrate, vertebrate and plant species. As a kind of AMPs, penaeidins play important roles in innate immunity of shrimp. In this study, two penaeidin homologues termed FmPEN3 and FmPEN5 were cloned and identified from Fenneropenaeus merguiensis for the first time. The complete open reading frames (ORFs) of FmPEN3 and FmPEN5 were 216 bp and 240 bp, encoding 71 and 79 amino acids, respectively. Both FmPEN3 and FmPEN5 contain an N-terminal proline-rich domain (PRD) and a C-terminal cysteine-rich domain (CRD). The genome structure of FmPEN3 and FmPEN5 genes both consist of 2 exons and 1 intron. qPCR analysis showed that FmPEN3 was constitutively expressed but FmPEN5 transcripts were found only in hemocytes, gills, epidermis, nerve and pyloric cecum. The FmPEN3 and FmPEN5 expression were responsive to Vibrio parahaemolyticus and Micrococcus lysodeikticus infection and their transcription levels were downregulated by RNAi silencing of the transcription factors FmDorsal and FmRelish. In addition, recombinant proteins of FmPEN3 (rFmPEN3) and FmPEN5 (rFmPEN5) were successfully expressed in E. coli. The antibacterial assays revealed that rFmPEN3 and rFmPEN5 could inhibit the growth of M. lysodeikticus but only rFmPEN5 could inhibit the growth of V. parahaemolyticus in vitro. In summary, the results presented in this study indicated the functions of FmPEN3 and FmPEN5 played in anti-bacterial immunity of F. merguiensis, providing some insights into the function of AMPs in shrimp.