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Glutamate production from aerial nitrogen using the nitrogen-fixing bacterium Klebsiella oxytoca.

ABSTRACT: Glutamate is an essential biological compound produced for various therapeutic and nutritional applications. The current glutamate production process requires a large amount of ammonium, which is generated through the energy-consuming and CO₂-emitting Haber-Bosch process; therefore, the development of bio-economical glutamate production processes is required. We herein developed a strategy for glutamate production from aerial nitrogen using the nitrogen-fixing bacterium Klebsiella oxytoca. We showed that a simultaneous supply of glucose and citrate as carbon sources enhanced the nitrogenase activity of K. oxytoca. In the presence of glucose and citrate, K. oxytoca strain that was genetically engineered to increase the supply of 2-oxoglutarate, a precursor of glutamate synthesis, produced glutamate extracellularly more than 1 g L^-1 from aerial nitrogen. This strategy offers a sustainable and eco-friendly manufacturing process to produce various nitrogen-containing compounds using aerial nitrogen.

SUBMITTER: Yoshidome D

PROVIDER: S-EPMC11009414 | biostudies-literature | 2024 Apr

REPOSITORIES: biostudies-literature

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Glutamate production from aerial nitrogen using the nitrogen-fixing bacterium Klebsiella oxytoca.

Yoshidome Daisuke D Hidaka Makoto M Miyanaga Toka T Ito Yusuke Y Kosono Saori S Nishiyama Makoto M

Communications biology 20240411 1

Glutamate is an essential biological compound produced for various therapeutic and nutritional applications. The current glutamate production process requires a large amount of ammonium, which is generated through the energy-consuming and CO<sub>2</sub>-emitting Haber-Bosch process; therefore, the development of bio-economical glutamate production processes is required. We herein developed a strategy for glutamate production from aerial nitrogen using the nitrogen-fixing bacterium Klebsiella oxy ...[more]

PMID: 38605181

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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).

Project description:Differences in genome size and gene content are among the most important signatures of microbial adaptation and genome evolution. Here, we investigated the patterns of genome variation among strains of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti. Using the sequenced strain Rm1021 as a reference, the genome size and gene content variations were analyzed among ten diverse natural strains, through pulse field gel electrophoresis (PFGE) and whole-genome microarray hybridizations. Our PFGE analysis showed a genome size range of 6.45-7.01Mbp, with the greatest variation arising from the pSymA replicon, followed by that of pSymB. No observable size difference was evident among the chromosomes. Consistent with this pattern of size differences, 41.2% of ORFs on pSymA were variably absent/present, followed by 12.7% on pSymB, and 3.7% on the chromosome. However, the percentages of ORFs that were variably duplicated were more evenly distributed among the three replicons, 11.0%, 16.5% and 15.3% respectively for ORFs on pSymA, pSymB and the chromosome. Among the 10 strains, the percentages of absent ORFs ranged from 1.51% to 6.35% and those of duplicated ORFs ranged from 0.27% to 8.56%. Our analyses showed that host plants, geographic origins, multilocus enzyme electrophoretic types, and replicon sizes had little influence on the distribution patterns of absent or duplicated ORFs. The proportions of ORFs that were either variably absent/present or variably duplicated differed greatly among the functional categories, for each of the three replicons as well as for the whole genome. Interestingly, we observed positive correlations among the three replicons in their numbers of absent ORFs as well as the numbers of duplicated ORFs, consistent with coordinated gene gains/losses in this important bacterium in nature. microarray:Sm6kOligo A total of 12 strains were included for the microarray analyses in this study. The 12 strains included two reference strains and ten other natural strains. Reference strain Rm1021 was the strain with a completely sequenced genome, from which the whole genome microarray was based on (Krol et al. 2004). The second reference strain RmF909 was derived from strain Rm1021 but with a deletion of 575 ORFs located on the megaplasmid pSymB (genomic location 106724-735431, Charles et al. 1991). The two reference strains were used for positive and negative controls in our microarray hybridization experiments and for establishing threshold values for determining whether an ORF is present or absent among natural strains.

Dataset Information

Glutamate production from aerial nitrogen using the nitrogen-fixing bacterium Klebsiella oxytoca.

Publications

Glutamate production from aerial nitrogen using the nitrogen-fixing bacterium Klebsiella oxytoca.

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