Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca
Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca
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DOI：
10.1073/pnas.1120788109
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发表时间：
2012-05-01
影响因子：
11.1
通讯作者：
Voigt, Christopher A.
中科院分区：
文献类型：
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作者：
Temme, Karsten;Zhao, Dehua;Voigt, Christopher A.
与单一性状相关的细菌基因通常集中在基因组的一个连续单元中，称为基因簇。由于寄主调控的复杂性、冗余性和整合性，很难对许多基因簇进行遗传操作。我们开发了一种系统的方法，通过仅使用合成的、特征良好的部分自下而上地重建基因簇来完全指定基因簇的遗传学。这一过程消除了所有本地法规，包括未被发现的法规。首先，所有非编码DNA、调节蛋白和非必需基因都被移除。基本基因的密码子被改变，以产生与野生型(WT)基因尽可能不同的DNA序列。对重新编码的基因进行计算扫描，以消除内部调节。它们被组织成操纵子，并置于合成部分(启动子、核糖体结合位点和终止子)的控制下，这些合成部分由间隔区部分在功能上分开。最后，由遗传传感器和电路组成的控制器调节基因表达的条件和动态。我们将这种方法应用于一个与农业相关的基因簇，该基因簇编码了将大气中的N-2转化为氨的固氮途径。天然基因簇由7个操纵子中的20个基因组成，编码23.5kb的DNA。我们构建了一个与WT几乎没有DNA序列一致性的“重构”基因簇，并为其定义了每个遗传部分的功能。这项工作展示了合成生物学工具重写复杂生物功能的遗传学编码的潜力，以促进获取、工程和可转移性。
Bacterial genes associated with a single trait are often grouped in a contiguous unit of the genome known as a gene cluster. It is difficult to genetically manipulate many gene clusters because of complex, redundant, and integrated host regulation. We have developed a systematic approach to completely specify the genetics of a gene cluster by rebuilding it from the bottom up using only synthetic, well-characterized parts. This process removes all native regulation, including that which is undiscovered. First, all noncoding DNA, regulatory proteins, and nonessential genes are removed. The codons of essential genes are changed to create a DNA sequence as divergent as possible from the wild-type (WT) gene. Recoded genes are computationally scanned to eliminate internal regulation. They are organized into operons and placed under the control of synthetic parts (promoters, ribosome binding sites, and terminators) that are functionally separated by spacer parts. Finally, a controller consisting of genetic sensors and circuits regulates the conditions and dynamics of gene expression. We applied this approach to an agriculturally relevant gene cluster from Klebsiella oxytoca encoding the nitrogen fixation pathway for converting atmospheric N-2 to ammonia. The native gene cluster consists of 20 genes in seven operons and is encoded in 23.5 kb of DNA. We constructed a "refactored" gene cluster that shares little DNA sequence identity with WT and for which the function of every genetic part is defined. This work demonstrates the potential for synthetic biology tools to rewrite the genetics encoding complex biological functions to facilitate access, engineering, and transferability.