Abstract
A multifunctional transgenic Lactobacillus with probiotic characteristics and an ability to degrade β-glucan and phytic acid (phytate) was engineered to improve nutrient utilization, increase production performance and decrease digestive diseases in broiler chickens. The Bacillus subtilis WL001 endoglucanase gene (celW) and Aspergillus fumigatus WL002 phytase gene (phyW) mature peptide (phyWM) were cloned into an expression vector with the lactate dehydrogenase promoter of Lactobacillus casei and the secretion signal peptide of the Lactococcus lactis usp45 gene. This construct was then transformed into Lactobacillus reuteri XC1 that had been isolated from the gastrointestinal tract of broilers. Heterologous enzyme production and feed effectiveness of this genetically modified L. reuteri strain were investigated and evaluated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that the molecular mass of phyWM and celW was approximately 48.2 and 55 kDa, respectively, consistent with their predicted molecular weights. Endoglucanase and phytase activities in the extracellular fraction of the transformed L. reuteri culture were 0.68 and 0.42 U/mL, respectively. Transformed L. reuteri improved the feed conversion ratio of broilers from 21 to 42 days of age and over the whole feeding period. However, there was no effect on body weight gain and feed intake of chicks. Transformed L. reuteri supplementation improved levels of ash, calcium and phosphorus in tibiae at day 21 and of phosphorus at day 42. In addition, populations of Escherichia coli, Veillonella spp. and Bacteroides vulgatus were decreased, while populations of Bifidobacterium genus and Lactobacillus spp. were increased in the cecum at day 21.
Highlights
In some plant seeds, β-glucan is the main constituent of the cell wall and phytic acid is the major storage form of phosphorus [1,2]
Phytase and endoglucanase are rarely secreted in the digestive tracts of non-ruminant animals
The endoglucanase gene celW of B. subtilis WL001 (1.5 kb SpeI–XbaI fragment) and the phytase gene mature peptide phyWM of Aspergillus fumigatus WL002 (1.32 kb NotI–SacII fragment) were ligated together with a ribosome binding site rbs into the Lactobacillus expression vector pLEM4156, generating pLEM4159-cel/phy (Figure 1)
Summary
Β-glucan is the main constituent of the cell wall and phytic acid (phytate) is the major storage form of phosphorus [1,2]. They both have a powerful ability to absorb protein and minerals (including P, Ca2+, Cu2+, Fe2+, Mn2+, Zn2+) to form insoluble complexes or chelate, which may decrease the activity and diffusion of digestive enzymes, hinder absorption of nutrients and stimulate bacterial proliferation [3,4,5]. Phytase and endoglucanase are rarely secreted in the digestive tracts of non-ruminant animals. Many studies have demonstrated that supplementation with exogenous enzyme preparations can eliminate anti-nutritional factors and improve nutrient utilization and animal productive performance [9,10,11,12,13,14]
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