Discovery of a highly efficient TylF methyltransferase via random mutagenesis for improving tylosin production.

Macrolides are currently a class of extensively used antibiotics in human and animal medicine. Tylosin is not only one of the most important veterinary macrolides but also an indispensable material for the bio- and chemo-synthesis of new generations of macrolide antibiotics. Thus, improving its production yield is of great value. As the key rate-limiting enzyme catalyzing the terminal step of tylosin biosynthesis in Streptomyces fradiae (S. fradiae), TylF methyltransferase's catalytic activity directly affects tylosin yield. In this study, a tylF mutant library of S. fradiae SF-3 was constructed based on error-prone PCR technology. After two steps of screening in 24-well plates and conical flask fermentation and enzyme activity assay, a mutant strain was identified with higher TylF activity and tylosin yield. The mutation of tyrosine to phenylalanine is localized at the 139th amino acid residue on TylF (TylFY139F), and protein structure simulations demonstrated that this mutation changed the protein structure of TylF. Compared with wild-type protein TylF, TylFY139F exhibited higher enzymatic activity and thermostability. More importantly, the Y139 residue in TylF is a previously unidentified position required for TylF activity and tylosin production in S. fradiae, indicating the further potential to engineer the enzyme. These findings provide helpful information for the directed molecular evolution of this important enzyme and the genetic modification of tylosin-producing bacteria.

A High-Throughput Method Based on Microculture Technology for Screening of High-Yield Strains of Tylosin-Producing Streptomyces fradiae.

Tylosin is a potent veterinary macrolide antibiotic produced by the fermentation of Streptomyces fradiae; however, it is necessary to modify S. fradiae strains to improve tylosin production. In this study, we established a high-throughput, 24-well plate screening method for identifying S. fradiae strains that produce increased yields of tylosin. Additionally, we constructed mutant libraries of S. fradiae via ultraviolet (UV) irradiation and/or sodium nitrite mutagenesis. A primary screening of the libraries in 24-well plates and UV spectrophotometry identified S. fradiae mutants producing increased yields of tylosin. Mutants with tylosin yield 10% higher than the wild-type strain were inoculated into shake flasks, and the tylosin concentrations produced were determined by high-performance liquid chromatography (HPLC). Joint (UV irradiation and sodium nitrite) mutagenesis resulted in higher yields of mutants with enhanced tylosin production. Finally, 10 mutants showing higher tylosin yield were re-screened in shake flasks. The yield of tylosin A by strains UN-C183 (6767.64 ± 82.43 µg/ml) and UN-C137 (6889.72 ± 70.25 µg/ml) was significantly higher than that of the wild-type strain (6617.99 ± 22.67 µg/ml). These mutant strains will form the basis for further strain breeding in tylosin production.

Comparison of differentially expressed genes in longissimus dorsi muscle of Diannan small ears, Wujin and landrace pigs using RNA-seq.

Introduction: Pig growth is an important economic trait that involves the co-regulation of multiple genes and related signaling pathways. High-throughput sequencing has become a powerful technology for establishing the transcriptome profiles and can be used to screen genome-wide differentially expressed genes (DEGs). In order to elucidate the molecular mechanism underlying muscle growth, this study adopted RNA sequencing (RNA-seq) to identify and compare DEGs at the genetic level in the longissimus dorsi muscle (LDM) between two indigenous Chinese pig breeds (Diannan small ears [DSE] pig and Wujin pig [WJ]) and one introduced pig breed (Landrace pig [LP]). Methods: Animals under study were from two Chinese indigenous pig breeds (DSE pig, n = 3; WJ pig, n = 3) and one introduced pig breed (LP, n = 3) were used for RNA sequencing (RNA-seq) to identify and compare the expression levels of DEGs in the LDM. Then, functional annotation, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and Protein-Protein Interaction (PPI) network analysis were performed on these DEGs. Then, functional annotation, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and Protein-Protein Interaction (PPI) network analysis were performed on these DEGs. Results: The results revealed that for the DSE, WJ, and LP libraries, more than 66, 65, and 71 million clean reads were generated by transcriptome sequencing, respectively. A total of 11,213 genes were identified in the LDM tissue of these pig breeds, of which 7,127 were co-expressed in the muscle tissue of the three samples. In total, 441 and 339 DEGs were identified between DSE vs. WJ and LP vs. DSE in the study, with 254, 193 up-regulated genes and 187, 193 down-regulated genes in DSE compared to WJ and LP. GO analysis and KEGG signaling pathway analysis showed that DEGs are significantly related to contractile fiber, sarcolemma, and dystrophin-associated glycoprotein complex, myofibril, sarcolemma, and myosin II complex, Glycolysis/Gluconeogenesis, Propanoate metabolism, and Pyruvate metabolism, etc. In combination with functional annotation of DEGs, key genes such as ENO3 and JUN were identified by PPI network analysis. Discussion: In conclusion, the present study revealed key genes including DES, FLNC, PSMD1, PSMD6, PSME4, PSMB4, RPL11, RPL13A, ROS23, RPS29, MYH1, MYL9, MYL12B, TPM1, TPM4, ENO3, PGK1, PKM2, GPI, and the unannotated new gene ENSSSCG00000020769 and related signaling pathways that influence the difference in muscle growth and could provide a theoretical basis for improving pig muscle growth traits in the future.

Effects of substitution of soybean meal with rapeseed meal and glutamine supplementation on growth performance, intestinal morphology, and intestinal mucosa barrier of Qiandongnan Xiaoxiang Chicken.

OBJECTIVE: The present study was to evaluate the effects of different rapeseed meal substitution (RSM) and glutamine (Gln) supplementation on growth performance, intestine morphology, and intestinal mucosa barrier of broilers. METHODS: Four hundred and twenty Qiandongnan Xiaoxiang Chicken at 1 day of age with similar weight were chosen and were randomly assigned into 7 groups, consisting of 10 replicates per group and 6 broilers per replicate. Three groups were provided with diets separately containing 0%, 10%, and 20% RSM, and the other four groups were fed with diets separately supplemented with 0.5% and 1% Gln based on the inclusion of 10% and 20% RSM. At 21 and 42 days of age, 10 broilers per group were chosen to collect plasma and intestinal samples for further analysis. RESULTS: The results showed that 10% RSM decreased average daily feed intake (ADFI) and average daily weight gain (ADG) of broilers at 21 days of age (p<0.05). Furthermore, both ADFI and ADG of broilers at 21 and 42 days of age were decreased by 20% RSM, while feed conversion ratio (FCR) was increased (p<0.05). Besides, 10% RSM resulted in lower intestinal villus height and the ratio of villus height to crypt depth, deeper crypt depth (p<0.05), combined with the lower mRNA expressions of occludin, claudin-1, and zonula occludens-1 (ZO-1) in broilers at 21 days of age (p<0.05). Similar results were also observed in broilers at 21 and 42 days of age fed with 20% RSM. However, 1% Gln improved the growth performance of broilers fed with 10% and 20% RSM (p<0.05), ameliorated intestine morphology and elevated mRNA expressions of occludin, claudin-1 and ZO-1 (p<0.05). CONCLUSION: In conclusion, the increasing inclusion of RSM resulted in more serious effects on broilers, however, 1.0% Gln could reverse the negative effects induced by the inclusion of RSM.

The relationship between MHC-peptide interaction and resistance to virus in chickens.

INTRODUCTION: The MHC-peptide interaction has a subtle influence on host resistance to virus. This paper aims to study the relationship between MHC-peptide interaction and MHC-related virus-resistance. METHODS: By 3D homology modeling, the structure of chicken BF2 molecule BF2*0201 (PDB code: 4d0d) was studied and compared with the known structures of BF2 molecule BF2*0401 (PDB code: 4e0r) to elucidate the characteristics of BF2*0201-binding antigenic peptides. RESULTS: The results show that due to the amino acid difference between the two binding groove of 4e0r and 4d0d, the size of the binding groove of the two are 1130 ų and1380 ų respectively, indicating the amino acid species that 4e0r binding peptide has lower selectivity than 4d0d; and because of large side chain conformation of Arg (especially Arg111) of 4e0r replaced by small side chain Tyr111 of 4d0d, the volume of central part of the binding groove of 4d0d is obviously larger than that of 4e0r, indicating that the restrictive of binding antigenic peptides for 4d0d is narrower than that of 4e0r; and on account of the chargeability of the binding groove of the two are different, namely the binding groove chargeability of 4e0r (strong positive polarity) and 4d0d (weak negative polarity). CONCLUSION: There are generally more peptides presented by the BF2 of B2 haplotype than by that of B4 haplotype, leading to more resistance of B2 than that of B4 to virus.

Dietary guanidineacetic acid supplementation ameliorated meat quality and regulated muscle metabolism of broilers subjected to pre-slaughter transport stress by metabolomics analysis.

Pre-slaughter transport stress could induce multiple comprehensive variations in physiological and metabolic parameters of broilers. However, the entire metabolomics of pre-slaughter transport stress and supplementation of exogenous energy regulatory substances on broilers is still poorly understood. The metabolome characteristics of broilers subjected to 3 h pre-slaughter transport stress combined with 1,200 mg/kg guanidinoacetic acid (GAA1,200) supplementation were analyzed using gas chromatography-mass spectrometry (GC-MS) in this study. The results showed that, compared to the control group (no transport), 3 h pre-slaughter transport stress (T3h) decreased creatine (Cr), phosphocreatine (PCr) and adenosine triphosphate (ATP), and increased adenosine diphosphate (ADP), adenosine monophosphate (AMP) and the ratio of AMP to ATP in pectoralis muscle (PM) of broilers by high performance liquid chromatography (HPLC) analysis. However, GAA1,200 supplementation reversed the negative effects induced by 3 h pre-slaughter transport stress. Besides, GAA1,200 supplementation elevated mRNA expression of creatine transporter in PM. Our metabolomics approaches demonstrated that 38 and 48 significant metabolites were separately identified between the control group and T3h group, and T3h group and 3 h pre-slaughter transport stress combined with GAA1,200 supplementation group using the standard of variable importance in the projection values >1 and P < 0.05. Among these, the metabolites involved in amino acid metabolism (alanine, glycine, serine, threonine, cysteine , methionine, phenylalanine, tyrosine, and tryptophan), oxidative stress (3-methylhistidine, 1-methylhistidine and glutathione), non-protein amino acid (citrulline) metabolism, and energy metabolism (Cr, PCr, sarcosine, and glycocyamine) were confirmed through pathway enrichment analysis, which could be chosen as suitable candidate targets for further analysis of the effects of exogenous energy substances on broilers subjected to transport stress.

Study on the contrast of the MHC-peptide interaction of B2/B21 haplotype and MHC-related virus resistance in chickens.

INTRODUCTION: Three-dimensional (3D) structures of MHC class I exert some influence by the MHC-peptide interaction over host resistance to the virus. The thesis aims at studying the connection between MHC-peptide interaction of B2/B21 haplotype and MHC-related resistance to the virus. METHODS: The structure of chicken MHC class I BF2*0201 from B2 haplotype was studied and contrasted with that of BF2*2101 from B21 haplotype by using DNAMAN and PyMol software. RESULTS: The amino acid difference resulted in the difference in size and changeability of the binding groove of the two, resulting in different choices on the binding polypeptide. 3bew's (the crystal structure of BF2*2101 bound to peptide RV10) small side chain His111 replaces the short side chain Tyr111 of 4cvx (the crystal structure of BF2*0201 bound to peptide YL9), and the very small amino acid of Ser69 and Ser97 make the middle of the 3bew's binding groove become apparently broad and bound restrictive of amino acid smaller. Moreover, due to the specific amino acids-Arg9, Asp24, and Asp73 of 4cvx and Arg9, Asp24, and His111 of 3bew, the effect of the polypeptide and the binding groove differ between the two, and 3bew tends to bind polypeptides with negatively charged amino acids, but the large space in the middle can also accommodate other amino acids. Contrasted with the binding groove characteristic of 4cvx, it can be said that the selectivity of 3bew is higher than that of 4cvx in the amino acid type of the binding polypeptide, so the B21 haplotype has more host resistance to the virus than that of the B2 haplotype in chicken. CONCLUSION: There are usually various kinds of peptides presented by the BF2*2101 molecules of B21 haplotypes, resulting in resistance to pathogenic microorganisms, such as Rous sarcoma virus and/or Marek's disease virus. These findings may have an important theoretical foundation for screening of virus antigen, vaccine design, and genetic resistance breeding.

Transcriptional profiling of buffalo mammary gland with different milk fat contents.

Milk fat is the most important energy substance in milk and contributes to its quality and health benefits. Water buffalo milk is well known for its high milk quality with higher fat contents compared with cattle milk. Dehong buffalo is a unique local swamp breed in Yunnan Province with higher milk fat and excellent milk quality which provides a good model for the investigation of the molecular mechanisms of milk fat deposition. In this study, we used RNA-seq to obtain mammary tissue transcriptomics of buffalo with different milk fat phenotypes including high(H), medium (M)and low (L) fat content groups. Comparative analyses of buffalo among three groups yielded differentially expressed genes (DEGs). Analyzing the number of different genes among H_VS_L, H_VS_M, and M_VS_L showed the same expression pattern between H_VS_M. The increasing expression levels of genes including CSN1S1, BTN1A1, LALBA, ALDH1L2, SCD and MUC15, and down-regulated expression levels of genes containing CCL2, CRABP2, ADTRP, CLU and C4A in H_VS_L and M_VS_L were found. GO and KEGG enriched pathways revealed these DEGs involved in milk protein and fat as well as immune response. The findings would enhance the understanding of the interplay between the milk composition and immune response, which suggests that the immune capacity should be considered when we tried to improve the milk quality.

Plant-Derived Polysaccharides Regulated Immune Status, Gut Health and Microbiota of Broilers: A Review.

In modern intensive breeding system, broilers are exposed to various challenges, such as diet changes and pathological environment, which may cause the increase in the incidence rate and even death. It is necessary to take measures to prevent diseases and maintain optimal health and productivity of broilers. With the forbidden use of antibiotics in animal feed, polysaccharides from plants have attracted much attention owing to their lower toxicity, lower drug resistance, fewer side effects, and broad-spectrum antibacterial activity. It had been demonstrated that polysaccharides derived from plant exerted various functions, such as growth promotion, anti-inflammation, maintaining the integrity of intestinal mucosa, and regulation of intestinal microbiota. Therefore, the current review aimed to provide an overview of the recent advances in the impacts of plant-derived polysaccharides on anti-inflammation, gut health, and intestinal microbiota community of broilers in order to provide a reference for further study on maintaining the integrity of intestinal structure and function, and the related mechanism involved in the polysaccharide administration intervention.

Guanidino-Acetic Acid: A Scarce Substance in Biomass That Can Regulate Postmortem Meat Glycolysis of Broilers Subjected to Pre-slaughter Transportation.

The different substances in biomass can regulate the metabolism and reproduction of broilers. Guanidino-acetic acid (GAA) is a natural feed additive that showed a potential application in dietary for broilers, while its amount is scarce in biomass. The objective of the present study was to investigate the effects of dietary supplemented with GAA on muscle glycolysis of broilers subjected to pre-slaughter transportation. A total of 160 Qiandongnan Xiaoxiang chickens were randomly assigned into three treatments, including a basal control diet without GAA supplementation (80 birds) or supplemented with 600 mg/kg (40 birds) or 1,200 mg/kg (40 birds) GAA for 14 days. At the end of the experiment, the control group was equally divided into two groups, thus resulting in four groups. All birds in the four groups aforementioned were separately treated according to the following protocols: (1) no transport of birds of the control group fed with the basal diet; (2) a 3-h transport of birds of the control group fed with the basal diet; (3) a 3-h transport of birds fed with diets supplemented with 600 mg/kg GAA; and (4) a 3-h transport of birds fed with diets supplemented with 1,200 mg/kg GAA. The results demonstrated that 3-h pre-slaughter transport stress increased corticosterone contents and lowered glucose contents in plasma (P < 0.05), decreased pH24 h (P < 0.05), and resulted in inferior meat quality evidenced by elevating the drip loss, cooking loss, and L∗ value (P < 0.05). Meanwhile, 3-h pre-slaughter transport stress decreased the contents of Cr and ATP in muscle (P < 0.05) and elevated the ratio of AMP:ATP and the glycolytic potential of muscle (P < 0.05). Moreover, 3-h pre-slaughter transport resulted in a significant elevation of mRNA expressions of LKB1 and AMPKα2 (P < 0.05), as well as the increase in protein abundances of LKB1 phosphorylation and AMPKα phosphorylation (P < 0.05). However, 1,200 mg/kg GAA supplementation alleviated negative parameters in plasma, improved meat quality, and ameliorated postmortem glycolysis and energy metabolism through regulating the creatine-phosphocreatine cycle and key factors of AMPK signaling. In conclusion, dietary supplementation with 1,200 mg/kg GAA contributed to improving meat quality via ameliorating muscle energy expenditure and delaying anaerobic glycolysis of broilers subjected to the 3-h pre-slaughter transport.

Effects of dietary hydroxyproline on collagen metabolism, proline 4-hydroxylase activity, and expression of related gene in swim bladder of juvenile Nibea diacanthus.

This study was conducted to investigate the effects of dietary hydroxyproline (Hyp) on tissue collagen level, proline 4-hydroxylase (P4H) activity as well as transcript levels of COL1As (COL1A1 and COL1A2) and P4Hαs (P4Hα(I), P4Hα(II), and P4Hα(III)) in juvenile Nibea diacanthus. A total of 450 fishes were randomized to six equal groups and fed the diet with graded supplementary Hyp-0, 5, 10, 15, 20, and 25 g kg-1 of dry matter for 8 weeks. Results showed that fish fed diets with 10 g kg-1 Hyp had significantly higher acid-soluble collagen (ASC) and total collagen (TC) concentrations in swim bladder than fish fed with the other diets (P < 0.05). The activity of P4H in liver and swim bladder showed a similar trend, showing first increase and then decrease with increasing dietary Hyp (P < 0.05). The mRNA expression of COL1As in swim bladder and muscle were significantly higher than those in the liver and intestines. Meanwhile, with increasing dietary Hyp, the relative expression of COL1As genes in swim bladder showed a similar pattern with the TC concentrations of swim bladder, increased significantly initially followed by a decrease. Increased dietary Hyp content corresponded with significant decrease in the mRNA level of P4Hαs in swim bladder. These results indicated that the dietary Hyp promotes the collagen accumulation of swim bladder to some extent, and the promoting action may be related to the expression of COL1As. The optimum supplement of dietary Hyp was estimated from TC of swim bladder with piecewise regression analysis to be 9.66 g kg-1.

Selenium Attenuates Staphylococcus aureus Mastitis in Mice by Inhibiting the Activation of the NALP3 Inflammasome and NF-κB/MAPK Pathway.

Mastitis is one of the most important diseases affecting the dairy industry in the world, and it also poses a great threat to human food safety. In this study, we explored whether selenium can inhibit the activation of the NALP3 inflammasome and NF-κB/MAPK pathway to achieve anti-inflammatory effects. Sixty BALB/c female mice were randomly divided into three groups according to diets of different selenium concentrations (high, normal, and low). After 90 days, mice fed the same selenium concentration were randomly divided into two smaller groups, one of which was inoculated with Staphylococcus aureus and the other injected with saline as a control. Through histopathologic examination staining, western blot, qPCR, and ELISA, the results showed that with increasing selenium concentrations, the expression levels of IL-1ß, TNF-α, NALP3, caspase-1, and ASC were decreased in mouse mammary tissue. Therefore, this study revealed that selenium can attenuate S. aureus mastitis by inhibiting the activation of the NALP3 inflammasome and NF-κB/MAPK pathway.

Sterol regulatory element binding protein-1: Molecular cloning, tissue distribution and gene expression level in response to nutritional regulation in mud crab, Scylla paramamosain.

In the present study, SREBP-1 cDNA was cloned from the hepatopancreas of mud crab (Scylla paramamosain) and characterized by performing rapid-amplification of cDNA ends. The 3361bp long full-length cDNA encodes a polypeptide with 1039 amino acids. Tissue distribution analysis revealed that SREBP-1 transcripts were widely distributed in various organs, with higher mRNA levels in the eyestalk and cranial ganglia. Further, expression level of SREBP-1 mRNA were up-regulated in proportion to the replacement of dietary fish oil (FO) with soybean oil (SO). These results may contribute to better understanding of the long-chain polyunsaturated fatty acids (LC-PUFA) biosynthetic pathway and regulation mechanism in mud crab.

Cloning, tissue distribution and nutritional regulation of a fatty acyl Elovl4-like elongase in mud crab, Scylla paramamosain (Estampador, 1949).

In this report, the full-length cDNA of fatty acyl Elovl4-like elongase was cloned from the hepatopancreas of Scylla paramamosain by rapid-amplification of cDNA ends (RACE). To the best of our knowledge, this is the first report of Elovl4-like elongase in crustaceans. The full-length cDNA of Elovl4-like was 1119bp, which included a 5'-terminal untranslated region (UTR) of 58bp, a 3'-terminal UTR of 44bp and an open reading frame (ORF) of 1017bp encoding a polypeptide of 338 amino acids. Tissue distribution analysis revealed that Elovl4-like transcripts are widely distributed in various organs, with high mRNA levels in the hepatopancreas and cranial ganglia. Further, Elovl4-like transcriptional levels in hepatopancreas were up-regulated in proportion to the replacement of dietary fish oil (FO) with soybean oil (SO). The result showed that Elovl4-like transcripts increased about 0.83 and 1.12-fold respectively when SO constituted 80% and 100% of total oil (P<0.05). These results may contribute to better understanding of the long-chain polyunsaturated fatty acids (LC-PUFA) biosynthetic pathway and regulation mechanism in this species.

Molecular cloning, mRNA expression and nutritional regulation of a Δ6 fatty acyl desaturase-like gene of mud crab, Scylla paramamosain.

Fatty acyl desaturases (Fads) are critical enzymes in the pathways for the biosynthesis of the highly unsaturated fatty acids (HUFA). Here we report on the molecular cloning, tissue expression and nutritional regulation of a Δ6 fatty acyl desaturase-like (Δ6 Fad-like) gene from mud crab, Scylla paramamosain. The full-length cDNA was 1973bp, with a 201bp of 5'-UTR, a 443bp of 3'-UTR, and an ORF of 1329bp that encoded a protein of 442 amino acids. Bioinformatics analysis showed that the deduced peptide sequence possessed the typical features of the microsomal Fads, including N-terminal cytochrome b5 domain containing the heme-binding motif (H-P-G-G), three histidine-rich boxes and three membrane-spanning regions. Sequence comparison revealed that the predicted protein had a high percentage identity (>53%) with Δ6 Fads from other crustacean species. The tissue distribution of mud crab Δ6 Fad-like mRNA was found predominantly in hepatopancreas, with lower expression levels in all other tissues. Quantitative real-time PCR showed that the Δ6 Fad-like transcriptional levels in hepatopancreas gradually increased with the increased replacement of dietary fish oil (FO) by soybean oil (SO). The replacement ratio of FO by SO up to 60%, 80%, and 100% were significantly up-regulated by about 2.40-fold, 2.99-fold and 3.02-fold compared with that in the control group (100% FO) respectively (P<0.05). These results may contribute to better understanding the HUFA biosynthetic pathway and regulation mechanism in this species.