Enzymatically prepared alginate oligosaccharides improve broiler chicken growth performance by modulating the gut microbiota and growth hormone signals

Background Alginate oligosaccharide(AOS)holds great potential as a novel feed supplement in farm animals.However,the effects of AOS on chicken health and the underlying mechanisms are not fully understood.This study aimed to optimize the enzymatic preparation of AOS by using bacterial alginate lyases expressed in yeast,investigate the effects of the prepared AOS on the growth performance and gut health of broiler chickens,and reveal the underlying mechanisms.Results Five alginate lyases from bacteria were cloned into Pichia pastoris GS115 and the alginate lyase PDE9 was expressed at relatively high yield,activity and stability in P.pastoris.Animal trials were carried out using 3201-day-old male Arbor Acres broilers(four groups;8 replicates/group×10 chicks/replicate)receiving either a basal diet or the same diet supplemented with 100,200 and 400 mg/kg PDE9-prepared AOS for 42 d.The results showed that dietary supplementation of 200 mg/kg AOS displayed the highest activity in promoting the birds’ADG and ADFI(P<0.05).AOS ameliorated the intestinal morphology,absorption function and barrier function,as indicated by the enhanced(P<0.05)intestinal villus height,maltase activity,and the expression of PEPT,SGLT1,ZNT1,and occludin.AOS also increased serum insulin-like growth factor-1,ghrelin(P<0.05),and growth hormone(P<0.1).Moreover,the concentrations of acetate,isobutyrate,isovalerate,valerate,and total SCFAs in cecum of birds fed AOS were significantly higher than the control birds(P<0.05).Metagenomic analysis indicated that AOS modulated the chicken gut microbiota structure,function,and microbial interactions and promoted the growth of SCFAs-producing bacteria,for example,Dorea sp.002160985;SCFAs,especially acetate,were found positively correlated with the chicken growth performance and growth-related hormone signals(P<0.05).We further verified that AOS can be utilized by Dorea sp.to grow and to produce acetate in vitro.Conclusions We demonstrated that the enzymatically produced AOS effectively promoted broiler chicken growth performance by modulating the chicken gut microbiota structure and function.For the first time,we established the connections among AOS,chicken gut microbiota/SCFAs,growth hormone signals and chicken growth performance.

P_(luxI)mutants with different promoting period and their application for quorum sensing regulated protein expression

Quorum sensing(QS)system can dynamically control the expression of proteins along with the cell growth.The promoting period of QS system has been little focused on until now.In this study,a self-induced dynamic regulated expression(SIDRE)system was constructed in Escherichia coli.To enable the system suitable for the expression of enzymes,promoter engineering was used to obtain P_(luxI)mutants.To test the SIDRE system,alginate lyase AL493 and esterase Est7 were used as target protein for expression.The enzyme activity of alginate lyase and esterase reached 96.38%and 106.71%of the control strains containing the T7 promoter.In high-density fermentation,the activity of alginate lyase expressed by the SIDRE system with P_(luxI)(T-38C)as promoter was 4.34-fold of that expressed by the T7 promoter.Therefore,the P_(luxI)mutants with different promoting periods and/or different strengths show great potential in both laboratory and industrial scale for protein expression.

Evolving strategies for marine enzyme engineering:recent advances on the molecular modification of alginate lyase

Alginate,an acidic polysaccharide,is formed byβ-D-mannuronate(M)andα-L-guluronate(G).As a type of polysaccharide lyase,alginate lyase can efficiently degrade alginate into alginate oligosaccharides,having potential applications in the food,medicine,and agriculture fields.However,the application of alginate lyase has been limited due to its low catalytic efficiency and poor temperature stability.In recent years,various structural features of alginate lyase have been determined,resulting in modification strategies that can increase the applicability of alginate lyase,making it important to summarize and discuss the current evidence.In this review,we summarized the structural features and catalytic mechanisms of alginate lyase.Molecular modification strategies,such as rational design,directed evolution,conserved domain recombination,and non-catalytic domain truncation,are also described in detail.Lastly,the application of alginate lyase is discussed.This comprehensive summary can inform future applications of alginate lyases.

Cloning,expression and improvement of catalytic activity of alginate lyase by site-directed mutation

Alginate lyase mainly produces active alginate oligosaccharides(AOS)by degrading alginate viaβ-elimination process.In this study,the Pseudoalteromonas sp.Alg6B alginate lyase-encoding gene alg6B-7 from polysaccharide lyase(PL)-7 family was successfully cloned,sequenced,expressed in Escherichia coli.Based on rational design and amino acid sequence alignment of the alginate lyase from various sources,four positive mutants were obtained.The specific enzyme activities of four mutants I62A,A99K,V132S,and L157T were 38.84%,42.85%,75.8%and 51.83%higher than that of the wild enzyme,respectively.The K_(cat)/K_(m) values of the four mutants were both increased,and the catalytic efficiency of V132S was 1.92-fold higher than that of the wild enzyme,especially.The rational design that was employed in this study achieved the dramatic improvement of catalytic activity,which may provide the application potential in industrial production.