Development of an amylolytic Lactobacillus plantarum silage strain expressing the Lactobacillus amylovorus alpha-amylase gene

Abstract

An amylolytic LactobaciUus plantarum silage strain with the starch-degrading ability displayed by Lactobacilus amylovorus was developed. An active fragment of the gene coding for ca-amylase production in L. amylovorus was cloned and integrated into the chromosome of the competitive inoculant strain L. plantarum Lp8O at the cbh locus. The a-amylase gene fragment was also introduced into L. plantarum Lp8O on an autoreplicative plasmid. Both constructions were also performed in the laboratory strain L. plantarum NCIB8826. All four recombinant strains secreted levels of amylase ranging from 23 to 69 U/liter, compared with 47 U/liter for L. amylovorus. Secretion levels were higher in L. plantarum NCIB8826 than in L. plantarum Lp8O derivatives and were higher in recombinant strains containing autoreplicative plasmids than in the corresponding integrants. The L. plantarum Lp8O derivative containing the L. amylovorus a-amylase gene fragment integrated into the host chromosome secreted at-amylase to a level comparable to that of L. amylovorus and was stable over 50 generations of growth under nonselective conditions. It grew to a higher cell

Heterologous protein secretion byLactobacillus plantarumusing homologous signal peptides

“…plantarum NCIMB8826, which exhibits efficient secretion compared with other Lact. plantarum strains (Fitzsimons et al 1994;Hols et al 1997). In addition, the genome is known (Kleerebezem et al 2003), making the SP of this bacterium easily accessible for genetic manipulation.…”

Comparative Characterization of Complete and Truncated Forms of Lactobacillus amylovorus α-Amylase and Role of the C-Terminal Direct Repeats in Raw-Starch Binding

“…Expression and secretion. Since L. plantarum is easily electroporable (31) and had already been used for efficient L. amylovorus ␣-amylase gene expression and secretion (8,17), it was chosen as the host for transformation of the two ␣-amylase constructs, pLPCR2-3 and pLPCR2-3⌬B/X. Despite identical promoter and signal sequences, AmyA⌬ was expressed at a higher level of amylase than AmyA.…”

New trends and opportunities in the development and use of inoculants for silage

“…Several groups are working at developing LAB strains with the ability to degrade polysaccharides. Fitzsimons et al [69] transferred a gene coding for amylase activity from L. amylovarus to strains of L. plantarum by either integration to the chromosome or to an autoreplicative plasmid. The recombinant strains exhibited stable amylase activity and grew well in pure cultures in media containing starch.…”