Refactoring the nitrogen fixation gene cluster fromKlebsiella oxytoca

Abstract

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.

Design-based re-engineering of biosynthetic gene clusters: plug-and-play in practice

“…When refactoring a biosynthetic gene cluster, most of the genes in the final construct will be the same as in the original gene cluster, but the overall architecture and codon usage will be greatly modified to enable effective heterologous expression [10] (Figure 2). Having a precise annotation of the original sequence (and reliable sequence information, preferably verified by re-sequencing) is crucial to be able to accurately remove all native regulation, such regulatory genes, promoters, ribosomal binding sites (RBSs) and small RNAs.…”

“…Changing the codon usage of the open reading frames themselves has the dual function of boosting translational efficiency in the heterologous host and removing unknown regulatory elements that were hidden in the coding sequence. The new sequence should again be scanned for known internal regulatory elements or undesirable secondary structures that may have been created accidentally [10]. Depending on the host, it might also be advantageous to change the start codon: for example, for streptomycetes it has been shown that genes starting with Overview of the process of detecting and prioritizing gene clusters for molecular characterization and refactoring.…”

“…In the study by Temme et al [10], the refactored gene cluster from K. oxytoca was redesigned to form four new operons, each under the control of its own promoter and framed by a terminator sequence. Within an operon, each gene had its own RBS and was separated from the neighboring genes by an insulator sequence.…”

“…The final construct is assembled and transferred to the chosen host for heterologous expression. Adapted from Temme et al [10 ].…”

“…Intriguingly, the process of refactoring can also be applied to entire gene clusters. This was pioneered by Temme et al [10 ], who refactored the entire 23.5-kb/20-gene nitrogen fixation cluster from Klebsiella oxytoca, redesigning the operon structures of the gene cluster and replacing all regulatory elements by synthetic regulatory parts that had been characterized and shown to work in the target host organism, orthogonally from its native regulation. The work of Watanabe et al [11] has shown that the replacement of native promoters by synthetic ones can indeed be used to obtain successful heterologous expression of known compounds.…”