title: Functional optimization of gene clusters by combinatorial design and assembly.
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.
authors.affiliation: Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Smanski
authors.affiliation: Electrical and Computer Engineering Department, Boston University, Boston, Massachusetts, USA.
authors.forename: Swapnil
authors.initials: S
authors.lastname: Bhatia
authors.affiliation: Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Dehua
authors.initials: D
authors.lastname: Zhao
authors.affiliation: Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: YongJin
authors.initials: Y
authors.lastname: Park
authors.affiliation: 1] Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] Broad Technology Labs, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Lauren
authors.initials: L
authors.lastname: B A Woodruff
authors.affiliation: Broad Technology Labs, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Georgia
authors.initials: G
authors.lastname: Giannoukos
authors.affiliation: Broad Technology Labs, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Dawn
authors.initials: D
authors.lastname: Ciulla
authors.affiliation: Broad Technology Labs, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Michele
authors.initials: M
authors.lastname: Busby
authors.affiliation: Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Johnathan
authors.initials: J
authors.lastname: Calderon
authors.affiliation: Broad Technology Labs, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Nicol
authors.affiliation: 1] Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] Broad Technology Labs, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: D Benjamin
authors.initials: DB
authors.lastname: Gordon
authors.affiliation: Electrical and Computer Engineering Department, Boston University, Boston, Massachusetts, USA.
authors.forename: Douglas
authors.initials: D
authors.lastname: Densmore
authors.affiliation: 1] Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] Broad Technology Labs, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Voigt
mesh_terms: D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D041121:Klebsiella oxytoca / Q000235:genetics* / Q000502:physiology; D005810:Multigene Family; D009586:Nitrogen Fixation / Q000235:genetics*; D009591:Nitrogenase / Q000235:genetics; D009876:Operon / Q000235:genetics; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
chemical_list: D009591:Nitrogenase