Multi-omics Analyses Of The Deinococcus Radiodurans Chassis Expressing A Nitrogen-fixing Module

In nature,only some prokaryotic microorganisms are able to fix nitrogen to ammonium under energetic,nitrogen-free and anaerobic conditions using nitrogenase systems,while the vast majority of organisms,especially all eukaryotes,do not possess this nitrogen-fixing capacity.In Gram-negative bacteria such as Klebsiella oxytoca,Azotobacter vinelandii and Pseudomonas stutzeri as well as in Gram-positive bacteria such as Paenibacillus polymyxa,nitrogen-fixing genes exist mainly in the form of gene clusters,a natural structurally rigorous and functionally complete nitrogen-fixing module.After nearly 50 years of exploration,researchers have successfully transferred nitrogen-fixing modules into the chassis of various prokaryotic microorganisms such as Escherichia coli and Pseudomonas fluorescens and obtained artificial recombinant strains with certain nitrogenase activity,but so far,no eukaryotic organisms with autonomous nitrogen-fixing ability have been obtained.The reasons for this situation are that the regulation of nitrogen-fixing module expression in chassis organisms such as epigenetic modifications are not suitable,the energy supply of nitrogen fixation reaction is not sufficient,and the lack of oxygen protection of nitrogenase are the key limiting factors affecting the establishment of artificial nitrogen fixation system,especially the eukaryotic nitrogen fixation system.Deinococcus radiodurans R1 is a Gram-positive extremophilic model microbial strain,and genome-wide analysis indicates that its evolutionary status is between eukaryotic and prokaryotic,especially the ability of the bacterium to grow under anaerobic and microaerobic conditions,so it can be used as a model chassis organism for the assembly and commissioning of nitrogen-fixing modules.Currently,neither epigenetic modifications of the nitrogen-fixing module nor its heterologous expression in extreme microorganisms have been reported.In this study,we constructed a recombinant Deinococcus radiodurans R1 as a chassis and introduced a nitrogen-fixing module from Gram-positive Paenibacillus polymyxa,and systematically analyzed the expression characteristics of the nitrogen-fixing module in the recombinant bacteria.The main results obtained are as follows:1.The nitrogen-fixing cluster(nif BHDKENXhes AV)from Paenibacillus polymyxa WLY78 was constructed onto the E.coli-D.radiodurans R1 shuttle plasmid p RADZ3 to obtain the recombinant plasmid p RADZ3-78,which was transformed into E.coli JM109 to obtain recombinant E.coli.The nitrogenase activity assay showed that the recombinant E.coli had nitrogenase activity under anaerobic conditions,and the nitrogenase activity level was about one-fifth of that of Pseudomonas stutzeri A1501,indicating that the recombinant plasmid had complete nitrogen fixation function and could be used as a functional module for transformation of Deinococcus radiodurans R1.2.The recombinant plasmid p RADZ3-78 was further introduced into the Deinococcus radiodurans R1 chassis and the recombinant Deinococcus radiodurans R78 was obtained,and the PCR assay confirmed the successful construction.The nitrogenase activity assay showed the recombinant Deinococcus radiodurans R78 had no detectable acetylene reduction activity under the different culture conditions tested.The expression of nitrogen fixation genes under six culture conditions with different ammonium and oxygen concentrations was determined by RT-PCR,and the results showed that all nine genes could transcribe m RNA under different NH₄⁺and O₂ conditions,and the transcription levels did not differ significantly under different ammonium and oxygen conditions,which is consistent with the characterization of the gene cluster as a?⁷⁰-dependent promoter,and also consistent with the results in E.coli chassis.Western Blot using antibodies to the nitrogenase iron protein Nif H showed that the recombinant strain R78 had no characteristic bands of Nif H protein under the six conditions tested.The above results indicate that the nitrogen-fixing module can be transcribed normally in this recombinant bacterium,but cannot transform the nitrogenase iron protein,resulting in the inability of the recombinant bacterium to fix nitrogen.3.In order to investigate the fitness of the expression regulation,energy synthesis or oxygen protection between Deinococcus radiodurans and the exogenous nitrogen-fixing modules,the whole gene transcriptome of recombinant Deinococcus radiodurans R78 was analyzed under high-nitrogen aerobic(100 m M NH₄⁺+21%O₂)and nitrogen-free anaerobic(0 m M NH₄⁺+0%O₂)conditions.The results showed that 193 genes were significantly up-regulated and 195 genes were significantly down-regulated in expression under nitrogen-free anaerobic conditions compared to high-nitrogen aerobic conditions,especially the expression of genes related to the tricarboxylic acid cycle pathway,nitrogen metabolism and iron-sulfur transport system were significantly down-regulated.Meanwhile,the expression of genes that might be involved in epigenetic modifications of the bacteria changed significantly,suggesting that the epigenetic modification system in this bacterium might have a regulatory effect on the expression of nitrogen fixing enzymes.4.To investigate whether there is an effect of the epigenetic modification system on the expression of nitrogenase in recombinant Deinococcus radiodurans R78,the methylation modifications at the whole genome DNA level of recombinant strain R78 were analyzed using the Pacbio platform.The results showed that the DNA modification type of recombinant strain R78 was mainly 4m C with more than1100 methylation sites under both high nitrogen aerobic and nitrogen-free anaerobic conditions,while6m A methylation modification was less,less than 100 sites.Compared with the high nitrogen aerobic conditions,the 4m C modification sites of recombinant strain R78 under nitrogen-free anaerobic conditions were reduced by 424,and the difference of 6m A methylation sites was not significant.The methylation modification characteristics of RNA of recombinant strain R78 under different ammonium oxygen conditions were preliminarily analyzed by dot-blot detection,and the results showed that m⁵C modification of RNA existed in recombinant strain R78,and the strongest m⁵C modification signal was observed at 0.5%O₂ concentration.It is tentatively speculated that the methylation modification of DNA and RNA in recombinant Deinococcus radiodurans R78 may be related to the expression of nitrogenase,and the specific mechanism deserves further study.In summary,the nitrogen fixation module was able to be transcribed normally in this recombinant bacterium,but the translation of the protein was affected.Further multi-omics analyses showed that the changes in the expression of genes of nitrogen metabolism,iron-sulfur transport,tricarboxylic acid cycle and other pathways as well as the epigenetic system of nucleic acids might be the limiting factors affecting the expression of nitrogenase in the recombinant strain.This study provides theoretical guidance for further nitrogen fixation module design and chassis optimization,and ultimately for the construction of an artificially efficient nitrogen fixation device.