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A hybrid nitrogenase with regulatory elasticity in Azotobacter vinelandii

ABSTRACT: Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCE Azotobacter vinelandii is a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineered A. vinelandii strains and nitrogenase variants can be modulated at the regulatory level. The engineered strain studied here responds by adjusting the expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts.

ORGANISM(S): Azotobacter vinelandii DJ

PROVIDER: GSE234075 | GEO | 2023/06/07

REPOSITORIES: GEO

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Project description:Photosynthetic microbes can produce the clean-burning fuel hydrogen using one of natureâ??s most plentiful resources, sunlight 1,2. Anoxygenic photosynthetic bacteria generate hydrogen and ammonia during a process known as biological nitrogen fixation. This reaction is catalyzed by the enzyme nitrogenase and consumes nitrogen gas, ATP and electrons 3. One bacterium, Rhodopseudomonas palustris, has a remarkable ability to obtain electrons from green plant-derived material 4,5 and to efficiently absorb both high and low intensity light energy to form ATP 6. Manipulating R. palustris or a similar organism to produce hydrogen commercially will require us to identify all its genes that contribute to hydrogen production and to understand how this process is regulated in cells. Here we describe mutant strains in which metabolism is redirected such that hydrogen production is uncoupled from nitrogen fixation. Our data indicate that three different single amino acid changes in the transcriptional regulator NifA each yielded strains that produced hydrogen even in the presence of the repressing nitrogen source ammonium and in the absence of specific inducing metabolic signals. We used the mutants to show that, in addition to nitrogenase genes, 18 genes outside of the nitrogenase gene cluster may contribute to hydrogen production. Some of these genes are likely involved in efficient ATP acquisition and in channeling electrons to nitrogenase for reduction of protons to molecular hydrogen. Our results demonstrate that photosynthetic bacteria can be genetically manipulated for sustained production of pure hydrogen in a variety of cultivation conditions in the absence of oxygen, nitrogen or other gases as long as light and an electron donor are supplied. Transcriptome profile of wild type (CGA009) growing photosynthetically in the presence of amonium an acetate was compare with that of 4 different mutants (CGA570, CGA571, CGA572 and CGA574). We did 2 biological replicates per strain.

Project description:Purpose: Klebsiella oxytoca M5a1 (previously Klebsiella pneumonia M5a1) is a principle model organism for free-living biological nitrogen fixation. This strain has been used for decades in the characterisation of nitrogenase function and the genetic regulation of nitrogen fixation physiology. Currently it represents a key model for synthetic biology and biotechnology approaches. This project involves a multi-omics approach to modelling nitrogen physiology in Klebsiella oxytoca M5a1, in order to inform rational genetic engineering for improved nitrogen fixation activity. Here we studied the transition from nitrogen replete (high NH4Cl) to nitrogen starved (NH4Cl run-out) conditions, the latter of which induce nif gene expression and synthesis of the nitrogenase enzyme. We mapped RNA-sequencing (Illumina) reads to our assembled M5aI genome in order to compare the transcriptome between nitrogen replete and nitrogen fixing conditions using differential gene expression (DEG) analysis. Methods: Total RNA was extracted from fixed M5a1 cell samples after culturing in nitrogen replete (10 mM NH4Cl; one replicate) or nitrogen starved (3 hours after complete run-out of 0.5 mM NH4Cl; two replicates) growth medium (NFDM). cDNA libraries were prepared for Illumina (NextSeq 500) sequencing, with a read length of 75 nt and a depth of 100 million reads. Following processing and quality control, sequence reads were mapped to a M5a1 genomic template (GenBank WGS accession JAFHKG010000000; BioSample SAMN17288411) and gene count normalisation performed (RPKM). Differential gene expression analysis was performed using the DEseq2 package utilising settings of regularized logarithm (rlog) normalisation of read counts, Wald hypothesis testing and p-value adjustment using a False Discovery Rate threshold of 0.05. Log2 fold changes between conditions were calculated for differentially expressed genes (DEGs) with an adjusted p value of <0.05. Results: Across the three samples, between 22.0-25.1 million reads (81.4-87.7%) were mapped to the genome, including 56.7-68.5% mapped to annotated genes. More than 50% of genes (2694/5279 genes) were differentially expressed (adjusted p-value <0.05) in the nitrogen starved condition with respect to the nitrogen replate condition. 925 genes were upregulated and 1117 genes downregulated by more than 2-fold. Of these, 1515 differentially expressed genes correspond to annotated proteins in the KEGG functional orthology (KO) database. The 20 nitrogen fixation (nif) genes were ranked among the 200 genes with highest calculated expression values (RPKM) in the nitrogen starved condition. Conclusions: Our study provides a detailed insight into the global gene expression profile associated with diazotrophic (nitrogen fixing) growth, revealing a dominant role for nif and other nitrogen regulatory genes in the adaptation to nitrogen stress, in addition to a large array of secondary genes indirectly associated with shifts in metabolism and growth phenotype (e.g. metabolic enzymes, gene expression machinery, transporters).

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A hybrid nitrogenase with regulatory elasticity in Azotobacter vinelandii

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