A rapid and versatile tool for genomic engineering in Lactococcus lactis
A rapid and versatile tool for genomic engineering in Lactococcus lactis
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DOI：
10.1186/s12934-019-1075-3
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发表时间：
2019-01-31
影响因子：
6.4
通讯作者：
Kong, Jian
中科院分区：
文献类型：
--
作者：
Guo, Tingting;Xin, Yongping;Kong, Jian
背景乳酸乳球菌（Lactococcus lactis）是从生理特性到工业开发利用最广泛的乳酸菌之一。近十年来，L.乳酸菌已被开发成用于生产生物活性化合物如酶、疫苗抗原和天然产物的细胞工厂。然而，其精确和高效的基因组编辑工具仍然需要使L。结果从6种重组酶中筛选出一种高活性重组酶RecT，该重组酶可介导单链DNA（ssDNA）与乳酸链球菌（L. lactis）的同源重组。乳酸菌染色体rpoB位点，利福平筛选效率为100%。为了筛选没有外部选择表型的突变体，使用CRISPR/Cas9系统进行反选择，产生效率为46%的upp突变体。通过优化携带CRISPR/Cas9系统的质粒的拷贝数和间隔区序列的长度，消除了recA、galK、hemN和noxD基因的脱靶效率。使用upp和galK基因座作为靶标证明了该优化工具进行顺序点突变的能力，效率提高> 75%。此外，在72 h内可高效地完成基因组DNA的缺失（50/100 bp）或插入（loxP序列，34 bp）。通过ssDNA重组工程与改进的CRISPR/Cas9反选择的组合进行乳酸菌基因组工程。该工具将简化用于评估基因功能或构建生物合成宿主的等基因菌株的生产。
BackgroundLactococcus lactis is one of the most extensively characterized lactic acid bacteria, from physiological traits to industrial exploitation. Since last decade, L. lactis has been developed into cell factories for the production of bioactive compounds such as enzymes, vaccine antigens and natural products. However, its precise and efficient genome editing tools is still required to make L. lactis more suitable candidate for engineered functionality.ResultsA high active recombinase, RecT of Enterococcus faecalis ATCC14506, was selected from six candidates and mediated homologous recombination between single-stranded DNA (ssDNA) and the L. lactis chromosomal rpoB locus with an efficiency of 100% after rifampin selection. To screen mutants without an externally selectable phenotype, the CRISPR/Cas9 system was used for counterselection, yielding an upp mutant with an efficiency of 46%. By optimization of the copy number of plasmid carrying the CRISPR/Cas9 system and the length of spacer sequence, the off-target efficiency of the recA, galK, hemN and noxD genes were eliminated. The ability of this optimized tool to perform sequential point mutation was demonstrated using the upp and galK gene loci as targets with improved efficiencies>75%. Moreover, seamless genomic DNA deletions (50/100bp) or insertion (a loxP site, 34bp) was efficiently accomplished within 72h.ConclusionsThe work provided a rapid, versatile and precise tool for L. lactis genomic engineering by combination of ssDNA recombineering with improved CRISPR/Cas9 counterselection. This tool will simplify the production of isogenic strains for assessment of gene function or construction of biosynthetic host.