Construction Of Lactobacillus Transforming Phytase And Endoglucanase Genes And Evaluation Of Its Feeding Effectiveness On Broilers

β-glucan and phytic acid (phytate) are two main anti-nutrition factors in the diet ofnon-ruminant animals, have acutely strong absorption and chelation ability to nutrients(protein, lipids, mineral ions). Meanwhile, non-ruminant animals (such as pigs and poultry)lack or rarely secrete enzymes to hydrolyze β-glucan and phytic acid (phytate), which resultsin low dietary utilization and environmental pollution. Anti-nutritional factors could beeliminated by supplementation with homologous exogenous enzyme preparations in actualproduction. However, these supplements increase feed cost substantially and are onlyeffective in the short term. On the other hand, many natural enzymes cannot withstand thehigh temperatures of the feed pelleting process. A feasible method to overcome theseproblems might therefore be the development of multifunctional probiotics with degradationactivity, achieved by introducing heterologous genes encoding hydrolase. The objective of thestudy is to screen the stains with high phytase or endogulcanase activity from the nature world,and screen the predominant and probiotic Lactobacillus from broilers’ gastrointestinal tract(GI) as host bacteria, then clone homologous phytase and endoglucanase genes intoLactobacillus to construct multifunctional transgenic Lactobacillus with phytase activity,endoglucanase activity and probiotic properties and also evaluation of its feedingeffectiveness on broilers administrated barley-wheat diet with low effective phosphorus levelto improve nutrient utilization, increase production performance, decrease digestive diseasesin broiler chickens, save feed cost and establish the foundation for succedent application ofthe transgenic Lactobacillus reuteri on animal production.The specific results are as follows:1. A bacteria with endoglucanase activity and a fungal with phytase activity werescreened from the nature world, identified that belong to Bacillus subtilis and Aspergillusfumigatus by observation of colonial morphology and stain as well as determination of theconserved sequence16SrDNA or18SrDNA, and named them as B. subtilis WL001and A.fumigatus WL002, respectively. For B. subtilis WL001, the optimal carbon source, nitrogensource, initial pH and incubation time of producing endoglucanase were wheat bran, peptone,6-7,24h, respectively, the optimal temperature and pH of endoglucanase reaction were60°C and5.5, respectively. For A. fumigatus WL002, the optimal carbon source, nitrogen source,initial pH and incubation time of producing phytase were wheat bran, peptone,5-6,24h,respectively, the optimal temperature and pH of phytase reaction were60°C and5.5-6.5,respectively.2. Endoglucanase gene was successfully cloned from B. subtilis WL001genome DNA,about1500bp, named celW, and obtained the Genbank accession number (No. KJ528404).celW accomplished solubility expression in Ecoil BL21(DE3), the protein molecular weightwas55kD, extracellular endoglucanase activity was1.18U/ml. The mature peptide ofphytase gene was also successfully cloned from A. fumigatus WL002genome DNA, about1320bp, named phyAW, and obtained the Genbank accession number (No. KJ528403).phyAW accomplished solubility expression in Ecoil Transetta (DE3), the protein molecularweight was48.2kD, extracellular phytase activity was0.64U/ml.3. Lactobacilli had the highest diversity in the crop and the lowest one in the cecum.Compared with the lower GI tract, more Lactobacilli were found in the upper GI tract. L.reuteri, L. johnsonii, L. acidophilus, L. crispatus, L. salivarius and L. aviarius were thepredominant Lactobacillus species and present throughout the GI tract of chickens. L. reuteriwas the most abundant Lactobacillus species. L. reuteri XC1had good probioticcharacteristics that would be a potential and desirable target for genetic engineering.4. The singal gene expression plasmid and the co-expression plasmid of endglucanaseand phytase genes based on the Ecoil-Lactobacillus shuttle plasmid pLEM415weresuccessfully constructed, and named as pLEM4157(cel), pLEM4158(phy) andpLEM4159-cel/phy, respectively. Obtained the transgenic Lactobacillus: L. reuteripLEM4157(cel) with endoglucanase activity (0.96±0.08U/ml), L. reuteri pLEM4158(phy)with phytase activity (0.51±0.13U/ml) and L. reuteri pLEM4159-cel/phy with endoglucanaseactivity (0.68±0.17U/ml) and phytase activity (0.42±0.05U/ml), which possessed the similaracid and bile salts tolerance with wild L. reuteri. At pH=2and3, their cell survival rate wasabout13%and21%, respectively, and the cell survival rate in the presence of0.3%and0.5%bile salt was about10%and0.3%, respectively.5. L. reuteri pLEM4158(phy) and L. reuteri pLEM4159-cel/phy supplementation of abarley-wheat-based diet improved the feed conversion ratio from21to42days of age, and L.reuteri pLEM4157(cel), L. reuteri pLEM4158(phy) and L. reuteri pLEM4159-cel/phyimproved over the whole feeding period, but did not improve body weight gain or feed intakeof chicks.6. L. reuteri pLEM4159-cel/phy improved the ash, calcium and phosphorus contents oftibiae at day21, L. reuteri pLEM4157(cel) only improved the phosphorus contents of tibiae at day21. The phosphorus and calcium levels of tibiae in chicks administered transformed L.reuteri had a tendency to increase at day21. L. reuteri pLEM4158(phy) improved thephosphorus contents of tibiae at day42.7. Transformed L. reuteri supplementation of a barley-wheat-based diet decreased thepH in duodenum jejunum, drip loss and cooking loss of muscles, improved the muscelstenderness of chinkens at day21and42, but did not effect the Carcass characteristic, only hada light decrease to abdominal fat.8. L. reuteri pLEM4159-cel/phy increased the villi height in duodenum of chickens atday21and42, L. reuteri pLEM4158(phy). L. reuteri pLEM4159-cel/phy and L. reuteripLEM4157(cel) decreased the crypt depth in duodenum and jejunum, increased the intestinalwell thickness in jejunum of chickens at day21, and also decreased the crypt depth in ileumand increased the villi height in duodenum of chickens at day42. L. reuteri pLEM4158(phy)and L. reuteri pLEM4159-cel/phy improved the villi height and width in ileum of chickens atday42.9. L. reuteri pLEM4159-cel/phy significantly decreased the counts of E. coli in cecum ofchickens at day21. L. reuteri pLEM4157(cel) decreased the counts of Veillonella spp. incecum of chickens at day21. The counts of E. coli, Veillonella spp. and B. vulgatus in thececum of chickens administered transformed L. reuteri had a tendency to decrease at day21,but the counts of Bifidobacterium genus and Lactobacillus spp had a tendency to increase atday21, and the counts of Enterococcus faecalis and Lactobacillus spp had a tendency toincrease at day42.In a word, this work obtained a bacterium (B. subtilis WL001) with endoglucanaseactivity and a fungal (A. fumigatus WL002) with phytase activity, also successfully clonedtwo objective genes, which were solubilitily expressed in Ecoil. L. reuteri was the mostabundant Lactobacillus species. L. reuteri XC1was a potential and desirable target for geneticengineering. This work obtained a multifunctional transgenic Lactobacillus with phytaseactivity, endoglucanase activity and probiotic properties. Recombinant L. reuteri tended toimprove the feed conversion ratio, tibiae properties, intestinal tract pH, intestinal morphologyand microflora population in cecum of broilers.