Organelle-dependent polyprotein designs enable stoichiometric expression of nitrogen fixation components targeted to mitochondria

Abstract

Introducing nitrogen fixation (nif ) genes into eukaryotic genomes and targeting Nif components to mitochondria or chloroplasts is a promising strategy for engineering nitrogen-fixing plants. A prerequisite for achieving nitrogen fixation in crops is stable and stoichiometric expression of each component in organelles. Previously, we designed a polyprotein-based nitrogenase system depending on Tobacco Etch Virus protease (TEVp) to release functional Nif components from five polyproteins. Although this system satisfies the demand for specific expression ratios of Nif components in Escherichia coli, we encountered issues with TEVp cleavage of polyproteins targeted to yeast mitochondria. To overcome this obstacle, a version of the Nif polyprotein system was constructed by replacing TEVp cleavage sites with minimal peptide sequences, identified by knowledge-based engineering, that are susceptible to cleavage by the endogenous mitochondrial-processing peptidase. This replacement not only further reduces the number of genes required, but also prevents potential precleavage of polyproteins outside the target organelle. This version of the polyprotein-based nitrogenase system achieved levels of nitrogenase activity in E. coli, comparable to those observed with the TEVp-based polyprotein nitrogenase system. When applied to yeast mitochondria, stable and balanced expression of Nif components was realized. This strategy has potential advantages, not only for transferring nitrogen fixation to eukaryotic cells, but also for the engineering of other metabolic pathways that require mitochondrial compartmentalization.

Keywords: mitochondrial-processing peptidase; nitrogenase; polyprotein; synthetic biology.

Fig. 1.

Engineering and assessment of the TEVp-based Nif polyprotein system in yeast mitochondria. (A) Schematic diagram of the TEVp-based nif constructs for expression in S. cerevisiae. Giant genes are highlighted in red color and symbol “ǒ” is used to represent dual TEVp sites (ENLYFQSENLYFQS). The selected marker Leu2 (indicated above in red) is used for auxotrophic selection of transformants. Constructs were integrated in the YNRCΔ9 locus on chromosome XIV of the yeast genome with ~600 bp 5′YNRCΔ9 and 3′YNRCΔ9 flanking regions as homology arms (Dataset S1). (B) Immunoblotting of Nif proteins from yeast carrying the TEVp-based Nif polyprotein system. Ec Nif, indicates protein samples prepared from E. coli cells carrying the reconstituted operon-based nif system (24). Mit Isolations, indicates protein samples prepared from mitochondrial extracts. HSP60 was used as an internal reference and a Coomassie blue stained PAGE gel was used as control for equal loading of each sample. Long-term exposure of these images is provided in SI Appendix, Fig. S3. (C) Growth curves of yeast strains carrying the TEVp-based Nif polyprotein system. Sc_535, is a yeast strain transformed with empty vector carrying the Leu2 selection marker (EV) assigned as a control. Sc_410 and Sc_411, are yeast strains carrying constructs pNG410 and pNG411 respectively. “Growth rate” represents the relative maximum growth rate of each strain. The maximum growth rate of strain Sc_535 was assigned as 100%. In the panel on the left, glucose was initially present at 2%. For the right hand-panel, 0.4% of glucose was used for pregrowth, and the final ratio of glucose and galactose was 1:4.

Fig. 2.

Screening for minimal MPP-processing sites. (A) Schematic diagram showing plasmid constructs and design procedure for testing the cleavage efficiency of reconstituted MPP in E. coli. GFP-RFP fusions linked by MPP cleavage sites were expressed from the σ⁵⁴-dependent P_nifH promoter from K. oxytoca and activated by NifA constitutively expressed from the P_tet promoter. MPP (α subunit labeled with HA-tag and β subunit labeled with His-tag) were controlled by inducible P_tac/lacO promoter. The diamond symbol represents the MPP sites. (B) Sequences of the designed MPP sites. “AA” is short for amino acid. Arginine residues, which may be important for MPP recognition are underlined. Flexible linker regions are highlighted in blue color. The red symbol “↓” represents the proposed processing site of MPP. Panels (C–F) show immunoblotting assays to determine the cleavage efficiency of the various MPP sites listed in (B). The symbol “−”, indicates the absence of the MPP expression module; “+”, the presence of the MPP expression module induced with 100 μM IPTG. (G) Assessment of the cleavage efficiency of selected MPP sites in mitochondria of S. cerevisiae. The “2×” symbol indicates that tandem MPP sites were used. HSP60 was used as an internal reference for mitochondrial specificity.

Fig. 3.

Assembly and characterization of the MPP-based Nif polyprotein system in E. coli. (A) Example of a dataset for nifHDK encoded polyproteins linked by different MPP cleavage sequences (listed in Fig. 2B). The acetylene reduction assay (ARA) was used to measure complementation by the nifHDK giant gene by the remaining nif genes in the operon-based system, either in the absence of MPP (orange bars, plasmid pKU7871) or presence of MPP (green bars, plasmid pG32) after induction with IPTG. WT, indicates complementation by the native nifHDKTY operon carrying the NifD-Y100Q substitution and this activity was assigned as 100% (24.2 ± 1.1 nmol C₂H₄/min/mg total protein). S9 and S10 indicate single copies of the S9 site (RGGRRAFHT) or the S10 site (RGGGRRAFHT) respectively. Tandem processing sites are indicated by 2 × S9 and 2 × S10, respectively. Red arrows indicate the unprocessed NifDK protein. (B) Summary data for the nifHDK, nifENB, nifUS, nifJVW, and nifFMY encoded polyproteins ^aAcetylene reduction activities restored by the corresponding native nif genes or operons were assigned as 100%. ^b2 × S10: indicates dual S10 sites (RGGGRRAFHT). ^c2 × S10S: indicates dual S10S sites (RGGGRRAFST). ^dData from Yang et al. (21). (C) Schematic diagram showing the process of assembly in which TEVp-based giant genes where replaced by MPP-based giant genes. Each ensemble was assigned a version number (Ver 1.0 to Ver 1.4, Ver 2.0, and Ver 2.1, respectively) listed on the left and analyzed by acetylene reduction. Symbols “ǒ”, “ǐǐ”, and “šš” are used to represent twofold TEVp sites, MPP S10 sites and S10S sites, respectively. In each case, ARA activities are expressed as a percentage of the activity exhibited by the TEVp-based polyprotein system (Ver 1.0), which was assigned as 100%. To detect activities of strains carrying Ver 1.1 to 1.4, both MPP and TEVp were expressed concomitantly (cotransformed with pBDS3668 plasmid). Error bars in panels (A–C) indicate the SD observed from at least two biological replicates. (D) Diazotrophic growth promoted by TEVp-based and MPP-based polyprotein systems in E. coli. WT represents the reconstituted operon-based nif system. EV represents empty vector (pBDS1549), used as a negative control. Ver1.0 and Ver 2.0 represent the TEVp- and MPP-based polyprotein system as shown in (C), respectively.

Fig. 4.

Assessment of MPP-based Nif polyprotein expression in yeast mitochondria. (A) Example of the NifHššDššK polyprotein expressed from the P_ScGAL1 promoter and targeted to mitochondria with the Su9 leader sequence in the yeast strain Sc_3682. Ec Nif indicates protein samples prepared from E. coli cells carrying the reconstituted operon-based nif system; Sc_535 is a yeast strain transformed with the empty vector pBDS535, used as a negative control. “Mit Isolations” indicates protein samples prepared from mitochondrial extracts. Image J software was used for protein quantification, and relative expression levels are shown in red (in parentheses as a percentage below lanes) (B) Schematic diagram showing parts used to build constructs pBDS3752 and pBDS3942. The only difference between these two constructs is the presence of promoter variants in pBDS3942 highlighted in red. Giant genes are highlighted in blue and the symbols “ĭĭ” and “šš” are used to represent dual MPP S10 and S10S sites respectively. The selection marker and integration site are the same as described in Fig. 1A. (C) Immunoblotting of yeast strains carrying MPP-based Nif polyprotein systems. One representative protein from each polyprotein was selected for the immunoblot assay. HSP60 was used as an internal reference and a Coomassie blue–stained PAGE gel was used as control for equal loading of each sample.

Fig. 5.

Engineering of the violacein and isobutanol biosynthesis pathways in mitochondria using the MPP-based polyprotein strategy in S. cerevisiae. (A) Schematic diagram showing the violacein biosynthesis pathway and gene arrangement in the polyproteins. (B) Petri dish experiment displaying the violet pigment biosynthesized by the MPP-based Vio polyprotein in yeast mitochondria. I: yeast strain transformed with the empty vector (pNG305). II: yeast strain transformed with the MPP-based vioAǐǐBǐǐE and vioDǐǐC polyprotein system carrying dual S10 sites (pNG297), in which five violacein biosynthesis genes were assembled as two polyproteins encoded by giant genes. III: yeast strain transformed with MPP-based vioAǐǐBǐǐE and vioDǐǐC polyprotein system carrying noncleavable S10 site variants (pNG299). The symbol “ǐǐ” represents the dual S10 site variant, in which the arginine residues at positions −2 and −3 were replaced by two alanine residues (RGGGAAAFSTRGGGAAAFST), to provide a negative control; IV: yeast strain transformed with the MPP-based vioAǐǐBǐǐEǐǐDǐǐC polyprotein system with tandem S10 sites (pNG300), in which five violacein biosynthesis genes were assembled as a giant gene encoding a single polyprotein. Immunoblotting (C) and solubility (D) analysis of Vio proteins extracted from the corresponding yeast strains indicated in (B). VioE and VioC proteins were labeled with hexahistidine and HA tags, respectively. “P” and “S” in blue color represent the insoluble and soluble fraction of proteins isolated from mitochondria respectively. “Nonspecific” indicates a protein present in yeast extracts that cross-reacts with the anti-HA antibody. “Mit Isolations” indicates protein samples prepared from mitochondrial extracts. HSP60 was used as internal reference and a Coomassie blue–stained PAGE gel was used as control for equal loading of each sample. (E) Schematic diagram showing the pathway genes and the combinatorial strategy employed to optimize isobutanol biosynthesis in yeast mitochondria using polyproteins. (F) Isobutanol production from MPP-based polyprotein combinations shown in (E). EV, indicates empty vector (plasmid pNG255), to assess the native level of isobutanol synthesized in yeast. Polyprotein combinations with the highest isobutanol production level are shown in red. Detailed information for each plasmid is provided in Dataset S1.