Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri.
Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri.
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DOI：
10.4161/bioe.21049
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发表时间：
2012-07
期刊：
影响因子：
4.9
通讯作者：
Britton RA
中科院分区：
文献类型：
--
作者：
Van Pijkeren JP;Neoh KM;Sirias D;Findley AS;Britton RA
Single-stranded DNA (ssDNA) recombineering is a technology which is used to make subtle changes in the chromosome of several bacterial genera. Cells which express a single-stranded DNA binding protein (RecT or Bet) are transformed with an oligonucleotide which is incorporated via an annealing and replication-dependent mechanism. By in silico analysis we identified ssDNA binding protein homologs in the genus Lactobacillus and Lactococcus lactis. To assess whether we could further improve the recombineering efficiency in Lactobacillus reuteri ATCC PTA 6475 we expressed several RecT homologs in this strain. RecT derived from Enterococcus faecalis CRMEN 19 yielded comparable efficiencies compared with a native RecT protein, but none of the other proteins further increased the recombineering efficiency. We successfully improved recombineering efficiency 10-fold in L. lactis by increasing oligonucleotide concentration combined with the use of oligonucleotides containing phosphorothioate-linkages (PTOs). Surprisingly, neither increased oligonucleotide concentration nor PTO linkages enhanced recombineering in L. reuteri 6475. To emphasize the utility of this technology in improving probiotic features we modified six bases in a transcriptional regulatory element region of the pdu-operon of L. reuteri 6475, yielding a 3-fold increase in the production of the antimicrobial compound reuterin. Directed genetic modification of lactic acid bacteria through ssDNA recombineering will simplify strain improvement in a way that, when mutating a single base, is genetically indistinguishable from strains obtained through directed evolution.
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影响因子：
5.6
作者：
Sawitzke JA;Costantino N;Li XT;Thomason LC;Bubunenko M;Court C;Court DL
通讯作者：
Court DL
DOI：
10.1073/pnas.121164898
发表时间：
2001-06-05
影响因子：
11.1
作者：
Ellis, HM;Yu, DG;Court, DL
通讯作者：
Court, DL
影响因子：
5.8
作者：
Guimaraes, VD;Gabriel, JE;Langella, P
通讯作者：
Langella, P
影响因子：
2.8
作者：
Sorvig, E;Mathiesen, G;Axelsson, L
通讯作者：
Axelsson, L
影响因子：
56.9
作者：
Steidler, L;Hans, W;Remaut, E
通讯作者：
Remaut, E