Use of the lambda Red-recombineering method for genetic engineering of Pantoea ananatis.
Use of the lambda Red-recombineering method for genetic engineering of Pantoea ananatis.
复制标题
使用 lambda Red 重组工程方法进行菠萝泛菌的基因工程。
DOI：
10.1186/1471-2199-10-34
复制
发表时间：
2009-04-23
影响因子：
--
通讯作者：
Mashko SV
中科院分区：
文献类型：
--
作者：
Katashkina JI;Hara Y;Golubeva LI;Andreeva IG;Kuvaeva TM;Mashko SV
假单胞菌是肠杆菌科细菌属的一员，是生物技术研究的一个新课题。近年来，其在L味精生产中的应用取得了令人印象深刻的进展。然而，由于缺乏在该细菌中快速构建直接突变的遗传工具，对该细菌的遗传和生物技术研究一直受到阻碍。λ红色重组工程技术以前是在大肠杆菌中开发的，用于其他几种细菌的基因失活，是一种快速构建精确基因组修改的高性能工具。在本研究中，λRed基因在南美白对虾中的表达被发现是高度毒性的。通过筛选，筛选出能抵抗λ红毒害作用的突变体。筛选出一株能同时表达λgam、bet和exo基因的突变菌株SC17(0)。使用该菌株，已经证明了基于PCRRed依赖整合的λ产生的DNA片段的多重重排快速引入泛藻基因组的程序，该片段的侧翼同源性最短为40bp。λ红色重组工程技术被成功地用于快速产生染色体修饰的特殊选育的水华螺受体株。建立了染色体DNA电转化的方法，用于在不同菌株之间转移显著突变。这些技术与依赖于λINT/XIS的选择标记的切除相结合，极大地加快了生产菌株的基础研究和构建。
Pantoea ananatis, a member of the Enterobacteriacea family, is a new and promising subject for biotechnological research. Over recent years, impressive progress in its application to L-glutamate production has been achieved. Nevertheless, genetic and biotechnological studies of Pantoea ananatis have been impeded because of the absence of genetic tools for rapid construction of direct mutations in this bacterium. The λ Red-recombineering technique previously developed in E. coli and used for gene inactivation in several other bacteria is a high-performance tool for rapid construction of precise genome modifications. In this study, the expression of λ Red genes in P. ananatis was found to be highly toxic. A screening was performed to select mutants of P. ananatis that were resistant to the toxic affects of λ Red. A mutant strain, SC17(0) was identified that grew well under conditions of simultaneous expression of λ gam, bet, and exo genes. Using this strain, procedures for fast introduction of multiple rearrangements to the Pantoea ananatis genome based on the λ Red-dependent integration of the PCR-generated DNA fragments with as short as 40 bp flanking homologies have been demonstrated. The λ Red-recombineering technology was successfully used for rapid generation of chromosomal modifications in the specially selected P. ananatis recipient strain. The procedure of electro-transformation with chromosomal DNA has been developed for transfer of the marked mutation between different P. ananatis strains. Combination of these techniques with λ Int/Xis-dependent excision of selective markers significantly accelerates basic research and construction of producing strains.
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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：
3.5
作者：
CHEREPANOV, PP;WACKERNAGEL, W
通讯作者：
WACKERNAGEL, W
影响因子：
3.5
作者：
BRUNSCHWIG, E;DARZINS, A
通讯作者：
DARZINS, A
DOI：
10.1073/pnas.94.7.2951
发表时间：
1997-04-01
影响因子：
11.1
作者：
Hill, SA;Stahl, MM;Stahl, FW
通讯作者：
Stahl, FW
影响因子：
3.2
作者：
KRISTENSEN, CS;EBERL, L;DELORENZO, V
通讯作者：
DELORENZO, V