Many genome sequences of useful industrial microorganisms have been completely sequenced. Based on the results of these genome sequences, functional analyses of microbial genomes have been carried out using comparative genomics and bioinformatics to define functions of new genes and metabolic pathways. These genomic analyses have also been performed in the Escherichia coli which is an important experimental and industrial organism and 620 essential genes and 3,126 nonessential genes have been identified. Of the nonessential genes, E. coli possesses transposable elements, phages, cryptic prophages, motility related genes, horizontally transferred genes, pseudogenes, gene remnants, and damaged operons that would not be needed in the strains designed for biotechnology purposes. These nonessential genes express many unnecessary gene products that represent potential contaminants and metabolic wastes entailed in producing unwanted products that could drive up the cost of product purification and are extra burdens on cell growth. In addition, the genetic instability of the microorganism owing to transposable elements is certainly a problem for producing useful biomaterials and rational strain improvement. Therefore, deletion of these nonessential genes allows us to improve the microorganism for biotechnology purposes. However, conventional methods for deleting many nonessential genes scattered along the E. coli genome have limitations that these methods require either the creation of targeting vectors or complex PCR experiments per each deletion. The need for numerous deletion experiments led us to develop a rapid and efficient deletion procedure.
Here, we have developed a powerful genome-engineering tool to delete the nonessential genes of E. coll. We have made two large pools of independent transposon mutants in E. coli using modified Tn5 transposons with a loxP site and two different selection markers and precisely mapped the chromosomal location of 1,000 of these transposons. By combining a mapped transposon mutation from each of the mutant pools into the same chromosome using phage P1 transduction and then excising the flanked genomic segment by Cre-mediated loxP recombination, we have obtained 33 E. coli deletion strains in which large genomic fragments (19-117 kb) were deleted and the total size of the deletions was 1.15 Mb (-25% of the E. coli genome). Some of these individual deletions were then combined into a single cumulative deletion strain (EGD 10) in which 637.4 kb (-15% of the E. coli genome) were deleted. This cumulative deletion strain exhibited similar growth rate comparable to that of wild type strain in LB broth, shows slightly elongated cell size and expresses highly some specific genes to complement some functions of the deleted genes. In addition, this genomic tool could be applicable to identify essential genes (b26I5 and serV) and mutually exclusive genes (sucAB and sucCD) in E. coli. This genomic tool can be directly applied to minimization of the E. coli genome, generating a simplified metabolic model that could be used for mathematical modeling of whole cell metabolism and the Tn5-mutant libraries and deletion strains can also be applied to improving industrial E. coli strains by restructuring metabolic and cellular pathways.
본 연구에서는 대장균내의 불필요한 유전자들을 빠르고 효과적으로 제거할 수 있는 새로운 유전체 제거방법을 개발하였다. 이러한 방법에서는 하나의 loxP site가 대장균 유전체 내에 고르게 삽입되어진 두 종류의 mutant libraries를 구축하였고, 여기에 P1 transduction과 Cre/loxP excision system을 이용하여 대장균 유전체의 불필요한 부분들을 쉽게 제거할 수 있었다. 이러한 방법을 이용하여 9-117 kb 정도의 유전체 부분들이 각각 제거되어진 33개의 대장균 유전체 제거균주들을 확보하였고, 각각의 유전체 제거부분들을 하나의 유전체내로 모아 637 kb의 대장균 유전체가 제거되어진 새로운 유전체 제거균주를 구축하였다. 이러한 균주는 Wild type 대장균과 유사한 성장곡선을 보이며 약간 길어진 세포모양을 한다. 또한 제거되어진 유전자들의 기능을 보완하기 위해 여러 paralog들이 높게 발현됨을 DNA chip analysis와 2-D gel analysis로 확인할 수 있었다. 이와 더불어 이러한 유전체 제거기술은 E. coli 유전체내에 존재하는 mutually exclusive genes 찾는데도 이용되어질 수 있다.
