Zhiru Tang

Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition

Horizontal gene transfer by transposition has been widely used for transgenesis in prokaryotes. However, conjugation has been preferred for transfer of large transgenes, despite greater restrictions of host range. We examine the possibility that transposons can be used to deliver large transgenes to heterologous hosts. This possibility is particularly relevant to the expression of large secondary metabolite gene clusters in various heterologous hosts. Recently, we showed that the engineering of large gene clusters like type I polyketide/nonribosomal peptide pathways for heterologous expression is no longer a bottleneck. Here, we apply recombineering to engineer either the epothilone (epo) or myxochromide S (mchS) gene cluster for transpositional delivery and expression in heterologous hosts. The 58-kb epo gene cluster was fully reconstituted from two clones by stitching. Then, the epo promoter was exchanged for a promoter active in the heterologous host, followed by engineering into the MycoMar transposon. A similar process was applied to the mchS gene cluster. The engineered gene clusters were transferred and expressed in the heterologous hosts Myxococcus xanthus and Pseudomonas putida. We achieved the largest transposition yet reported for any system and suggest that delivery by transposon will become the method of choice for delivery of large transgenes, particularly not only for metabolic engineering but also for general transgenesis in prokaryotes and eukaryotes.

Effects of dietary supplementation with an expressed fusion peptide bovine lactoferricin-lactoferrampin on performance, immune function and intestinal mucosal morphology in piglets weaned at age 21 d

Lactoferrin has antimicrobial activity associated with peptide fragments lactoferricin (LFC) and lactoferrampin (LFA) released on digestion. These two fragments have been expressed in Photorhabdus luminescens as a fusion peptide linked to protein cipB. The construct cipB-LFC-LFA was tested as an alternative to antimicrobial growth promoters in pig production. Sixty piglets with an average live body weight of 5.42 (sem 0.59) kg were challenged with enterotoxigenic Escherichia coli and randomly assigned to four treatment groups fed a maize-soyabean meal diet containing either no addition (C), cipB at 100 mg/kg (C+B), cipB-LFC-LFA at 100 mg/kg (C+L) or colistin sulfate at 100 mg/kg (C+CS) for 3 weeks. Compared with C, dietary supplementation with C+L for 3 weeks increased daily weight gain by 21 %, increased recovery from diarrhoea, enhanced serum glutathione peroxidase (GPx), peroxidase (POD) and total antioxidant content (T-AOC), liver GPx, POD, superoxide dismutase and T-AOC, Fe, total Fe-binding capacity, IgA, IgG and IgM levels (P < 0.05), decreased the concentration of E. coli in the ileum, caecum and colon (P < 0.05), increased the concentration of lactobacilli and bifidobacteria in the ileum, caecum and colon (P < 0.05), and promoted development of the villus-crypt architecture of the small intestine. Growth performance was similar between C+L- and C+CS-supplemented pigs. The present results indicate that LFC-LFA is an effective alternative to the feed antibiotic CS for enhancing growth performance in piglets weaned at age 21 d.

Effects of Chitosan on Intestinal Inflammation in Weaned Pigs Challenged by Enterotoxigenic Escherichia coli

The aim of this study was to investigate whether supplementation with chitosan (COS) could reduce diarrhea and to explore how COS alleviates intestinal inflammation in weaned pigs. Thirty pigs (Duroc×Landrace×Yorkshire, initial BW of 5.65±0.27) weaned at age 21 d were challenged with enterotoxigenic Escherichia coli during a preliminary trial period, and then divided into three treatment groups. Pigs in individual pens were fed a corn-soybean meal diet, that contained either 0 (control), 50 mg/kg chlortetracycline, or 300 mg/kg COS for 21 days. The post-weaning diarrhea frequency, calprotectin levels and TLR4 protein expression were decreased (P<0.05) in both the COS and chlortetracycline groups compared with control. Simultaneously, supplemental COS and chlortetracycline had no effect on the mRNA expression of TNF-α in the jejunal mucosa, or on the concentrations of IL-1β, IL-6 and TNF-α in serum. However, COS supplementation improved (P<0.05) the mRNA expression of IL-1β and IL-6 in the jejunal mucosa. The results indicate that supplementation with COS at 300 mg/kg was effective for alleviating intestinal inflammation and enhancing the cell-mediated immune response. As feed additives, chitosan and chlortetracycline may influence different mechanisms for alleviating inflammation in piglets.

Expression, Purification, and Antibacterial Activity of Bovine Lactoferrampin–Lactoferricin in Pichia pastoris

Bovine lactoferrampin (LFA) and bovine lactoferricin (LFC) are two antimicrobial peptides located in the N1 domain of bovine lactoferrin. The bactericidal activity of the fused peptide LFA–LFC is stronger than that of either LFA or LFC. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current study, the expression, purification, and antibacterial activity of LFA–LFC using the Pichia pastoris expression system are reported. The linearized expression vector pPICZaA–LFA–LFC was transformed into P. pastoris KM71 by electroporation, and positive colonies harboring the target genes were screened out and used for fermentation. The recombinant LFA–LFC peptide was purified via two-step column chromatography and identified by tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate that P. pastoris is a suitable system for secreting LFA–LFC. The fermentation supernate and the purified LFA–LFC show high antimicrobial activities. The current study is the first to report on the expression and purification of LFA–LFC in P. pastoris and may have potential practical applications in microbial peptide production.

Effects of dietary administering chitosan on growth performance, jejunal morphology, jejunal mucosal sIgA, occluding, claudin-1 and TLR4 expression in weaned piglets challenged by enterotoxigenic Escherichia coli

This study was conducted to investigate how chitosan (COS) affects intestinal mucosal barrier function and to further explain mechanisms of COS on growth performance. Thirty piglets, weaned at 21 days of age, were challenged with enterotoxigenic Escherichia coli during preliminary trial period. Three groups of Piglets in individual pens were fed a corn-soybean meal diet containing no addition, 50 mg/kg chlortetracycline, or 300 mg/kg COS for 21 days. Jejunal morphology and histology were analyzed under light microscope. The concentrations of occludin proteins were determined by western blot. Immunohistochemistry assays were used to determine secretory immunoglobulin (sIgA) level. Real-time PCR was used to detect Toll-like receptor 4 (TLR4) and Claudin-1 in jejunal mucosa. Feeding COS or chlortetracycline reduced (P<0.05) feed conversion ratio. Villus length, villus length/crypt depth, and goblet cells, were increased (P<0.05), but villus width and crypt depth were decreased (P<0.05) in both COS and chlortetracycline groups. Intraepithelial lymphocytes were higher (P<0.05) in the COS group than both chlortetracycline and control groups. Occludin protein expression was increased (P<0.01) in the COS group, but was decreased (P<0.05) in the chlortetracycline group. Expression of sIgA protein was higher (P<0.05) in the COS group than both control and chlortetracycline groups, however TLR4 mRNA expression was decreased (P<0.05) in both COS and chlortetracycline groups. There was no difference in expression of claudin-1 among the three groups. In conclusion, chitosan and the antibiotic have similar effects in promoting piglet growth and reducing intestinal inflammation, but different effects on intestinal mucosal barrier function. This indicates that chitosan can replace chlortetracycline as a feed additive for piglets.

High-level expression, purification and antibacterial activity of bovine lactoferricin and lactoferrampin in Photorhabdus luminescens

Bovine lactoferricin (LFC) and bovine lactoferrampin (LFA) are two active fragments located in the N(1)-domain of bovine lactoferrin. Recent studies suggested that LFC and LFA have broad-spectrum activity against Gram-positive and Gram-negative bacteria. To date, LFC and LFA have usually been produced from milk. We report here the high-level expression, purification and characterization of LFC and LFA using the Photorhabdus luminescens expression system. After the cipA and cipB genes were deleted by ET recombination, the expression host P. luminescens TZR(001) was constructed. A synthetic LFC-LFA gene containing LFC and LFA was fused with the cipB gene to form a cipB-LFC-LFA gene. To obtain the expression vector pBAD-cipB-LFC-LFA, the cipB-LFC-LFA gene was cloned on the L-arabinose-inducible expression vector pBAD24. pBAD-cipB-LFC-LFA was transformed into P. luminescens TZR(001). The cipB-LFC-LFA fusion protein was expressed under the induction of L-arabinose and its yield reached 12 mg L(-1) bacterial culture. Recombinant LFC-LFA was released from cipB by pepsin. The MIC of recombinant LFC-LFA toward E. coli 0149, 0141 and 020 was 6.25, 12.5 and 3.175 microg ml(-1), respectively.

Pyruvate is an effective substitute for glutamate in regulating porcine nitrogen excretion

This study was performed to determine if pyruvate, which acts as a critical intermediate in energy metabolism, can substitute the role of glutamate as a metabolic fuel and effectively reduce nitrogen excretion in pigs. First, the experiment in vitro was carried out to investigate the effects of culturing porcine small intestinal epithelial cell line with pyruvate on the oxidation. Then, barrows weighing 40 kg were used in the experiment investigating the changes of nitrogen balance in response to addition of pyruvate to low-protein diets. Last, barrows (40 kg), which were surgically fitted with permanent catheters in the mesenteric vein, portal vein, hepatic vein, and carotid artery, were used to investigate the effects of supplementing low-protein diets with calcium pyruvate on the net portal fluxes of amino acids (AAs) and the consumption of AAs in the liver. The results showed that culturing cells with sodium pyruvate significantly reduced the number of glutamate oxidation (P < 0.05). Addition of calcium pyruvate to low-protein diets significantly reduced urinary nitrogen excretion from 13.2 g/d (18.0% crude protein, CP) to 10.3 g/d (15.0% CP) or 7.80 g/d (13.5% CP) and total nitrogen excretion from 22.5 g/d (18.0% CP) to 17.8 g/d (15.0% CP) or 14.2 g/d (13.5% CP) (P < 0.05), without obviously negative effects on the nitrogen retention (P > 0.05). Addition of calcium pyruvate to low-protein diets significantly decreased essential AA consumption rate in the liver (P < 0.05). This diet modification reduced the net portal fluxes of NH3, glycine, and alanine, as well as urea production rate in the liver (P < 0.05). The results indicated that pyruvate is an effective substitute for glutamate as a supplement in low-protein diets, reducing porcine nitrogen excretion and nitrogen consumption.

Activation of Pyruvate Dehydrogenase by Sodium Dichloroacetate Shifts Metabolic Consumption from Amino Acids to Glucose in IPEC-J2 Cells and Intestinal Bacteria in Pigs

The extensive metabolism of amino acids (AA) as fuel is an important reason for the low use efficiency of protein in pigs. In this study, we investigated whether regulation of the pyruvate dehydrogenase kinase (PDK)/pyruvate dehydrogenase alpha 1 (PDHA1) pathway affected AA consumption by porcine intestinal epithelial (IPEC-J2) cells and intestinal bacteria in pigs. The effects of knockdown of PDHA1 and PDK1 with small interfering RNA (siRNA) on nutrient consumption by IPEC-J2 cells were evaluated. IPEC-J2 cells were then cultured with sodium dichloroacetate (DCA) to quantify AA and glucose consumption and nutrient oxidative metabolism. The results showed that knockdown of PDHA1 using siRNA decreased glucose consumption but increased total AA (TAA) and glutamate (Glu) consumption by IPEC-J2 cells ( P < 0.05). Opposite effects were observed using siRNA targeting PDK1 ( P < 0.05). Additionally, culturing IPEC-J2 cells in the presence of 5 mM DCA markedly increased the phosphorylation of PDHA1 and PDH phosphatase 1, but inhibited PDK1 phosphorylation ( P < 0.05). DCA treatment also reduced TAA and Glu consumption and increased glucose depletion ( P < 0.05). These results indicated that PDH was the regulatory target for shifting from AA metabolism to glucose metabolism and that culturing cells with DCA decreased the consumption of AAs by increasing the depletion of glucose through PDH activation.

Low-Protein Diets Decrease Porcine Nitrogen Excretion but with Restrictive Effects on Amino Acid Utilization

Reducing dietary crude protein (CP) intake effectively decreases nitrogen excretion in growing-finishing pigs but at the expense of poor growth when dietary CP content is reduced by ≥3%. In this study, we investigated the main disadvantages of low-protein diets supplemented with lysine, methionine, threonine, and tryptophan in pigs. First, changes in the nitrogen balance in response to differences in dietary CP content (18%, 15%, and 13.5%) were investigated in barrows (40 kg). Then, barrows (40 kg) surgically fitted with catheters in the mesenteric vein, portal vein, hepatic vein, and carotid artery were used to investigate changes in amino acid (AA) metabolism in the portal-drained viscera and liver in response to differences in dietary CP content. The results showed that low-protein diets reduced fecal and urinary nitrogen excretion ( P < 0.05) meanwhile resulted in significant decreases in nitrogen retention ( P < 0.05). Moreover, a reduction in the dietary CP content from 18% to 13.5% resulted in decreases in the net portal fluxes of NH3, glycine, and alanine as well as in the urea production in the liver ( P < 0.05), whereas their values as a percentage of nitrogen intake did not decline ( P > 0.05). The net portal fluxes of nonessential AA (NEAA) were reduced in the low-protein diet groups ( P < 0.05), while essential AA consumption in the liver increased ( P < 0.05). Thus, low-protein diets result in reductions in both nitrogen excretion and retention, and NEAA deficiency may be a major disadvantage of low-protein diets.