Using synthetic biology to increase nitrogenase activity (original) (raw)
Related papers
PLoS ONE, 2013
Biological nitrogen fixation is a complex process requiring multiple genes working in concert. To date, the Klebsiella pneumoniae nif gene cluster, divided into seven operons, is one of the most studied systems. Its nitrogen fixation capacity is subject to complex cascade regulation and physiological limitations. In this report, the entire K. pneumoniae nif gene cluster was reassembled as operon-based BioBrick parts in Escherichia coli. It provided ,100% activity of native K. pneumoniae system. Based on the expression levels of these BioBrick parts, a T7 RNA polymerase-LacI expression system was used to replace the s 54-dependent promoters located upstream of nif operons. Expression patterns of nif operons were critical for the maximum activity of the recombinant system. By mimicking these expression levels with variable-strength T7dependent promoters, ,42% of the nitrogenase activity of the s 54-dependent nif system was achieved in E. coli. When the newly constructed T7-dependent nif system was challenged with different genetic and physiological conditions, it bypassed the original complex regulatory circuits, with minor physiological limitations. Therefore, we have successfully replaced the nif regulatory elements with a simple expression system that may provide the first step for further research of introducing nif genes into eukaryotic organelles, which has considerable potentials in agro-biotechnology.
The roles of the nifW, nifZ and nifM genes of Klebsiella pneumoniae in nitrogenase biosynthesis
European journal of biochemistry / FEBS, 1989
Active Fe protein of nitrogenase was synthesised in a non-nitrogen fixing organism when Escherichia coli was transformed with a plasmid encoding only two nif-specific genes, nifH and nifM of Klebsiella pneumoniae. Hence proteins NifH and NifM are sufficient to produce active Fe protein in E. coli. K. pneumoniae strains carrying chromosomal nifW- and nifZ- mutations were constructed and shown to be significant C2H2-reducing activity and to grow on N-free plates. Nevertheless, derepressing cultures of the mutant strains had reduced levels of MoFe protein activity, and consequently significantly lower levels of nitrogenase activity, than the nif+ parent strain. NifW and NifZ therefore appear to be involved in the formation or accumulation of active MoFe protein, but are not essential for nitrogen fixation in K. pneumoniae under the conditions tested.
Characterization and analysis of nifH genes from Paenibacillus sabinae T27
Microbiological Research, 2012
Paenibacillus sabinae T27 (CCBAU 10202=DSM 17841) is a gram-positive, spore-forming diazotroph with high nitrogenase activities. Three nifH clusters were cloned from P. sabinae T27. Phylogenetic analysis revealed that NifH1, NifH2 and NifH3 cluster with Cyanobacterium. Each of the coding regions of nifH1, nifH2 and nifH3 from P. sabinae T27 under the control of the nifH promoter of Klebsiella pneumoniae could partially restore nitrogenase activity of K. pneumoniae nifH − mutant strain 1795, which has no nitrogenase activity. This suggests that the three nifH genes from P. sabinae T27 have some function in nitrogen fixation. RT-PCR showed that all three nifH genes were expressed under nitrogen-fixing growth conditions. Using promoter vectors which have promoterless lacZ gene, three putative promoter regions of nifH genes were identified.
State of the art in eukaryotic nitrogenase engineering
FEMS microbiology letters, 2018
Improving the ability of plants and plant-associated organisms to fix and assimilate atmospheric nitrogen has inspired plant biotechnologists for decades, not only to alleviate negative effects on nature from increased use and availability of reactive nitrogen, but also because of apparent economic benefits and opportunities. The combination of recent advances in synthetic biology and increased knowledge about the biochemistry and biosynthesis of the nitrogenase enzyme has made the seemingly remote and for long unreachable dream more possible. In this review, we will discuss strategies how this could be accomplished using biotechnology, with a special focus on recent progress on engineering plants to express its own nitrogenase.
2020
Paenibacillus durus strain ATCC 35681 T is a Gram-positive diazotroph that displayed capability of fixing nitrogen even in the presence of nitrate or ammonium. However, the nitrogen fixation activity was detected only at day 1 of growth when cultured in liquid nitrogen-enriched medium. The transcripts of all the nifH homologues were present throughout the 9-day study. When grown in nitrogen-deficient medium, nitrogenase activities occurred from day 1 until day 6 and the nifH transcripts were also present during the course of the study albeit at different levels. In both studies, the absence of nitrogen fixation activity regardless of the presence of the nifH transcripts raised the possibility of a post-transcriptional or post-translational regulation of the system. A putative SigA box sequence was found upstream of the transcription start site of nifB1, the first gene in the major nitrogen fixation cluster. The upstream region of nifB2 showed a promoter recognisable by SigE, a sigma factor normally involved in sporulation.
Proceedings of the National Academy of Sciences, 2020
Significance Stable expression of each component of the nitrogenase system in an active form is a prerequisite for engineering nitrogen fixation in eukaryotic cells. Mitochondria provide an oxygen-depleted environment for the expression of active nitrogenase in plants, but signal peptides are required to target nuclear encoded Nif proteins to this organelle. We demonstrate that one of the structural subunits of nitrogenase, NifD, is itself susceptible to cleavage by mitochondrial processing peptidases from a variety of plant origins, presenting a major challenge to engineering nitrogen fixation in mitochondria. To overcome this issue, we have engineered NifD variants that are resistant to cleavage and retain high levels of nitrogenase activity, thus providing a potential solution for engineering active MoFe protein in plants.
Promoter mutations that allow nifA-independent expression of the nitrogen fixation nifHDKY operon
Proceedings of the National Academy of Sciences, 1983
The nifHDKY operon of KlebsieUa pneumoniae encodes 'for structural polypeptides of nitrogenase and requires the nifA gene product for transcription. Mutations that allow transcription of the niJHDKY operon in absence of the nifA gene product were characterized in plasmids containing the regulatory region of nifHDKY and nifH fused in:phase to lacZ.-B-Galactosidase activity served as a measure for nifHWexpression. Most mutations were located in the nif regulatory region and included insertion sequence 2 (IS2) insertions, a sequence duplication, and a base substitution. In Escherichia coli, 3-pgalactosidase activity expressed from the mutant plasmids in the absence of MfA was 6-30% of the.nifA-activated, parental level. Expression from most mutant plasmids was further increased by nifA. InK. pneumoniae, IS2-containing plasmids expressed low levels of S-galactosidase and responded poorly, if at all, to activation by nifA, whereas expression from other mutant types was similar to that observed in E. coli. Nucleotide sequence analysis of two mutants indicated that sequences within 41 base pairs upstream to the nifH coding sequence were involved in nif-specific regulation. The results suggest that an inverted repeat element in this region, which could theoretically form.a cruciform structure in the DNA, is involved in the transcriptional control of the nifHDKY operon.
Microbiology, 2011
Rhizobia are a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes (Fix phenotype). Synthesis of the nitrogenase and its accessory components is under the transcriptional control of the key regulator NifA and is generally restricted to the endosymbiotic forms of rhizobia known as bacteroids. Amongst studied rhizobia, Sinorhizobium fredii strain NGR234 has the remarkable ability to fix nitrogen in association with more than 130 species in 73 legume genera that form either determinate, indeterminate or aeschynomenoid nodules. Hence, NGR234 is a model organism to study nitrogen fixation in association with a variety of legumes. The symbiotic plasmid pSfrNGR234a carries more than 50 genes that are under the transcriptional control of NifA. To facilitate the functional analysis of NifA-regulated genes a new transposable element, TnEKm-PwA, was constructed. This transposon combines the advantages of in vitro mutagenesis of cloned DNA fr...
Kinetics of nif Gene Expression in a Nitrogen-Fixing Bacterium
Journal of Bacteriology, 2014
Nitrogen fixation is a tightly regulated trait. Switching from N 2 fixation-repressing conditions to the N 2 -fixing state is carefully controlled in diazotrophic bacteria mainly because of the high energy demand that it imposes. By using quantitative real-time PCR and quantitative immunoblotting, we show here how nitrogen fixation ( nif ) gene expression develops in Azotobacter vinelandii upon derepression. Transient expression of the transcriptional activator-encoding gene, nifA , was followed by subsequent, longer-duration waves of expression of the nitrogenase biosynthetic and structural genes. Importantly, expression timing, expression levels, and NifA dependence varied greatly among the nif operons. Moreover, the exact concentrations of Nif proteins and their changes over time were determined for the first time. Nif protein concentrations were exquisitely balanced, with FeMo cofactor biosynthetic proteins accumulating at levels 50- to 100-fold lower than those of the structura...
Microbiology, 1984
Derepression of nitrogen fixation (nzf) genes in Klebsiella pneumoniae following transfer from NHi-sufficiency to N-free medium was preceded by rapid expansion of the guanosine 5'-diphosphate 3'-diphosphate (ppGpp) pool. When derepressed in N-free medium supplemented with glutamine (600 yg ml-l), expression from the nifH and nifL promoters, determined as P-galactosidase activity in nif: : lac merodiploid strains, was stimulated 7-fold and nitrogenase activity 26-fold; ppGpp did not accumulate, remaining at the levels found in NH2-repressed populations. The relaxed mutant K. pneumoniae relA40, which accumulates only very low levels of ppGpp, showed partial derepression of nitrogenase activity in the presence of glutamine, thus ppGpp is unlikely to be an effector of nifexpression. ATP and GTP levels were elevated under conditions where nif expression was enhanced, consistent with previous data suggesting that maintenance of ATP levels is a prerequisite for the expression of nifgenes in K. pneumoniae.