Refactoring the nitrogen fixation gene cluster from Klebsiella 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.

Fig. 1.

The process of refactoring a gene cluster. Top: WT K. oxytoca nitrogen fixation gene cluster. The genes are colored by function: blue (nitrogenase), green (cofactor biosynthesis, shading corresponds to operons), yellow (e^- transport), and gray (unknown). The thin arrows show the length and orientation of the seven operons, and a horizontal bar indicates overlapping genes. The recoded genes are shown as dashed lines. The symbols used to define the refactored cluster and controller are defined in Figs. 4 and 5, respectively.

Fig. 2.

The robustness of the nitrogen fixation pathway to changes in the expression of component proteins. (A) The pathway for nitrogenase maturation is shown and proteins are colored by function (Fig. 1). The metal clusters are synthesized by the biosynthetic pathway (23, 24). Nitrogen fixation catalyzed by the matured nitrogenase is shown with its in vivo electron transport chain. (B) The tolerance of nitrogenase activity to changes in the expression of component proteins are shown. Activity is measured via an acetylene reduction assay and the percentage compared with WT K. oxytoca is presented. WT operons are expressed from a P_tac promoter on a low-copy plasmid (SI Materials and Methods). The promoter activity is calculated as the output of the P_tac promoter at a given concentration of IPTG and compared with a constitutive promoter. The effect of not including NifY (-Y) and NifX (-X) are shown in red. (C) The comparison of the strength of WT (black) and synthetic (white) RBSs is shown. The RBSs were measured through an in-frame transcriptional fusion (−60 to +90) with mRFP. The strength is measured as the geometric average from a distribution of cells measured by flow cytometry (SI Materials and Methods). The synthetic RBSs of nifF and nifQ are not intended to match the WT measurement. Error bars represent the SD of at least three experiments performed on different days.

Fig. 3.

Converting to T7* RNAP control. (A) Nitrogenase activity is shown as a function of promoter strength for each refactored operon in respective K. oxytoca KO strains (ΔnifHDKTY, ΔnifENX, ΔnifJ, ΔnifBQ, ΔnifF, and ΔnifUSVWZM). Vertical dashed lines indicate strength of the mutant T7 promoter that controls each operon in the complete refactored gene cluster. (B) A controller plasmid decouples operon expression from the inducible promoter. A T7 RNAP variant (T7* RNAP) was designed to reduce toxicity. A set of four mutated T7 promoters were used to control the expression of each operon (part numbers and sequences for mutants 1–4 are listed in SI Materials and Methods). P_tac activity under 1 mM IPTG induction is indicated by a dashed horizontal line. (C) Nitrogenase activity is compared for each refactored operon under the control of the P_tac promoter at the optimal IPTG concentration (black) and the controller with 1 mM IPTG and expression controlled by different T7 promoters (white). The T7 promoters used are P_T7.WT for operons HDKY, EN, and J; P_T7.2 for operons BQ and USVWZM; and P_T7.3 for F. Error bars represent the SD of at least three experiments performed on different days.

Fig. 4.

Comprehensive schematic illustration for the complete refactored gene cluster and controller. Each of the 89 parts is represented according to the Synthetic Biology Open Language visual standard (www.sbolstandard.org), and the SynBERC Registry part number (registry.synberc.org) and part activity are shown. The full sequences of each plasmid have been deposited in GenBank (SBa_000534, JQ903614; SBa_000559, JQ903615; SBa_000560, JQ903616). The T7 promoter strengths are measured with monomeric red fluorescent protein and reported in REUs (Materials and Methods). Terminator strengths are measured in a reporter plasmid and reported as the fold reduction in monomeric red fluorescent protein (RFP) expression compared with a reporter without a terminator. The RBS strength is reported in as arbitrary units of expression from the induced P_tac promoter (1 mM IPTG) and a fusion gene between the first 90 nt of the gene and RFP. The nucleotide numbers for the plasmids containing the refactored cluster and controller are shown. The codon identity of each recoded gene compared with WT is shown as a percentage.

Fig. 5.

Regulation of the complete refactored gene cluster. (A) Nitrogenase activity for the three controllers are shown: IPTG-inducible, aTc-inducible, and IPTG ANDN aTc logic. The gas chromatography trace is shown for each, as well as the calculated percent of WT activity (7.4% ± 2.4%, 7.2% ± 1.7%, and 6.6% ± 1.7% respectively). SD is calculated by using data from at least two experiments performed on different days. (B) Incorporation of ¹⁵N into cell biomass is shown. Nitrogen fixation from N₂ gas by the refactored gene cluster was traced by using ¹⁵N₂ and measured by using isotope ratio mass spectrometry. Data are represented as the fraction of cellular nitrogen that is ¹⁵N. The SD represents two experiments performed on different days. (C) The effect of ammonia on regulation of nitrogenase expression is shown. Acetylene reduction traces shown with (red) and without (blue) addition of 17.5 mM ammonium acetate for WT cells (Left) and cells bearing synthetic nif system (Right). The synthetic system was induced by controller 1 using 1 mM IPTG and exhibited nitrogenase activity of 1.1% ± 0.5% and 6.1% ± 0.4% with and without ammonium acetate, respectively. (D) T7* RNAP expression of controller 1 corresponding to C is shown. Strains carrying controller 1 and an RFP reporter plasmid were characterized under 1 mM IPTG induction with or without addition of ammonium acetate.