DOI: 10.5061/dryad.mcvdnck2s
title: On the flexibility of the cellular amination network in E. coli

description: 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.
description: <strong>Isolation
 and sequence analysis of glut-aux ASS-<em>alaA</em>
 mutants</strong> The glut-aux strain +<em>alaA </em>was
 inoculated to OD<sub>600</sub> of 0.02 in<em>
 </em>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-<em>alaA</em> 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 2x10<sup>9</sup> cells of an overnight
 culture in LB medium supplied with streptomycin and chloramphenicol (to
 maintain pZ-ASS-<em>alaA</em> 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
 <em>E.coli</em> 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 &gt;50% prevalence in all mapped
 reads) and searched for regions with coverage deviating more than 2
 standard deviations from the global median
 coverage.
description: The sequencing data obtained from Novogene for all strains was
 analyzed using breseq. <br>The reference sequences were
 GENOME_SIJ488.fasta and pZ-ASS-alaA.fasta<br>The sequencing results
 for the strains are as follows:<br><br>Glutaux +alaA mutant 1
 files: <br>D14_FDSW202507757-1r_HCK2GDSXY_L4_2.fasta
 <br>D14_FDSW202507757-1r_HHGV7DSXY_L2_2.fasta<br><br>Glutaux +alaA mutant 2 files:<br>D15_FDSW202507757-1r_HCK2GDSXY_L4_2.fasta <br>D15_FDSW202507757-1r_HHGV7DSXY_L2_2.fasta<br><br>Glutaux +alaA parent files:<br>D19_FDSW202507762-1r_HCHWNDSXY_L3_2.fasta<br>D19_FDSW202507762-1r_HHGV7DSXY_L1_2.fasta<br><br>Differences between parent Glutaux +alaA parent, and Glutaux +alaA mutant 1 or Glutaux +alaA mutant 2 , respectively, were reported as mutations in the manuscript.