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Nat Biotechnol DOI:10.1038/nbt.3063

Functional optimization of gene clusters by combinatorial design and assembly.

Publication TypeJournal Article
Year of Publication2014
AuthorsSmanski, MJ, Bhatia, S, Zhao, D, Park, YJ, Woodruff, LBA, Giannoukos, G, Ciulla, D, Busby, M, Calderon, J, Nicol, R, D Gordon, B, Densmore, D, Voigt, CA
JournalNat Biotechnol
Volume32
Issue12
Pages1241-9
Date Published2014 Dec
ISSN1546-1696
KeywordsHigh-Throughput Nucleotide Sequencing, Klebsiella oxytoca, Multigene Family, Nitrogen Fixation, Nitrogenase, Operon, Promoter Regions, Genetic, Transcription, Genetic
Abstract

Large microbial gene clusters encode useful functions, including energy utilization and natural product biosynthesis, but genetic manipulation of such systems is slow, difficult and complicated by complex regulation. We exploit the modularity of a refactored Klebsiella oxytoca nitrogen fixation (nif) gene cluster (16 genes, 103 parts) to build genetic permutations that could not be achieved by starting from the wild-type cluster. Constraint-based combinatorial design and DNA assembly are used to build libraries of radically different cluster architectures by varying part choice, gene order, gene orientation and operon occupancy. We construct 84 variants of the nifUSVWZM operon, 145 variants of the nifHDKY operon, 155 variants of the nifHDKYENJ operon and 122 variants of the complete 16-gene pathway. The performance and behavior of these variants are characterized by nitrogenase assay and strand-specific RNA sequencing (RNA-seq), and the results are incorporated into subsequent design cycles. We have produced a fully synthetic cluster that recovers 57% of wild-type activity. Our approach allows the performance of genetic parts to be quantified simultaneously in hundreds of genetic contexts. This parallelized design-build-test-learn cycle, which can access previously unattainable regions of genetic space, should provide a useful, fast tool for genetic optimization and hypothesis testing.

URLhttp://dx.doi.org/10.1038/nbt.3063
DOI10.1038/nbt.3063
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/25419741?dopt=Abstract

Alternate JournalNat. Biotechnol.
PubMed ID25419741
Grant List / / Howard Hughes Medical Institute / United States