On the flexibility of the cellular amination network in E. coli

Ammonium (NH4+) is essential to generate the nitrogenous building blocks of life. It gets assimilated via the canonical biosynthetic routes to glutamate and is further distributed throughout metabolism via a network of transaminases. To study the flexibility of this network, we constructed an Escherichia coli glutamate auxotrophic strain. This strain allowed us to systematically study which amino acids serve as amine source and found that several amino acids complement the auxotrophy, either by producing glutamate via transamination reactions or by their conversion to glutamate. In this network, we identified aspartate transaminase AspC as a major connector between many amino acids and glutamate. Additionally, we extended the transaminase network by the amino acids β-alanine, alanine, glycine and serine as new amine sources and identified D-amino acid dehydrogenase (DadA) as an intracellular amino acid sink removing substrates from transaminase reactions. Finally, ammonium assimilation routes producing aspartate or leucine were introduced. Our study reveals the high flexibility of the cellular amination network, both in terms of transaminase promiscuity and adaptability to new connections and ammonium entry points.The sequencing data obtained from Novogene for all strains was analyzed using breseq. The reference sequences were GENOME_SIJ488.fasta and pZ-ASS-alaA.fastaThe sequencing results for the strains are as follows:Glutaux +alaA mutant 1 files: D14_FDSW202507757-1r_HCK2GDSXY_L4_2.fasta D14_FDSW202507757-1r_HHGV7DSXY_L2_2.fastaGlutaux +alaA mutant 2 files:D15_FDSW202507757-1r_HCK2GDSXY_L4_2.fasta D15_FDSW202507757-1r_HHGV7DSXY_L2_2.fastaGlutaux +alaA parent files:D19_FDSW202507762-1r_HCHWNDSXY_L3_2.fastaD19_FDSW202507762-1r_HHGV7DSXY_L1_2.fastaDifferences between parent Glutaux +alaA parent, and Glutaux +alaA mutant 1 or Glutaux +alaA mutant 2 , respectively, were reported as mutations in the manuscript.Isolation and sequence analysis of glut-aux ASS-alaA mutants The glut-aux strain +alaA was inoculated to OD600 of 0.02 in tube cultures of 4 mL M9 + 20mM glycerol + 5mM alanine. Cell growth was monitored during prolonged incubation at 37°C for 7-14 days. Within that time several cultures started to grow and reached an OD above 1.0. Cells were streaked out on LB plates with streptomycin (to maintain the pZ-ASS-alaA plasmid) by dilution streak to generate single colonies. Isolates were inoculated into tube cultures of 4 mL M9 + 20mM glycerol + 5mM alanine, and the ones which immediately grew were used in genome sequence analysis. Genomic DNA was extracted using the Macherey-Nagel NucleoSpin Microbial DNA purification Kit (Macherey-Nagel, Düren, Germany) from 2x109 cells of an overnight culture in LB medium supplied with streptomycin and chloramphenicol (to maintain pZ-ASS-alaA plasmid). Construction of (microbial short insert libraries) PCR-free libraries for single-nucleotide variant detection and generation of 150 bp paired-end reads on an Illumina HiSeq 3000 platform were performed by Novogene (Cambridge, UK). Reads were mapped to the reference genome of E.coli MG1655 (GenBank accession no. U00096.3) using the software Breseq (Barrick Lab, Texas) (Deatherage & Barrick, 2014). Using algorithms supplied by the software package, we identified single-nucleotide variants (with >50% prevalence in all mapped reads) and searched for regions with coverage deviating more than 2 standard deviations from the global median coverage