Genomics

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Transcriptomic analysis of homoserine-evolved MG1655 strain (wild type) and of homoserine -treated wild type, mutant deleted for thrL and mutant bearing thrL* allele


ABSTRACT: The growth of E. coli is inhibited by millimolar concentrations of L-homoserine, which may be problematic for the industrial production of this compound or for products derived from it. In this work, an adapted laboratory evolution (ALE) was applied, which resulted in the isolation of an E. coli strain at least 10-fold more tolerant to L-homoserine than the original (MG1655) strain. Only four genomic modifications were identified after genome sequencing of this evolved strain (designed 4E), including a 49 bp truncation starting from the codon stop of thrL and leading to a modified thrL locus carrying a thrL* allele encoding a 30 amino acids polypeptide, which is 9 amino acids more than the leader peptide encoded by thrL. Replacement of thrL with thrL* enabled the initial strain to be as or slightly more tolerant to L-homoserine than the evolved 4E strain, which is explained by the rapid metabolization of L-homoserine to threonine resulting from ThrL*-dependent transcriptional activation of the threonine thrABC operon. Interestingly, in 4E strain, L-homoserine degradation went beyond threonine degradation, as tdh and kbl encoding threonine degradation pathway II and genes of the glycine cleavage system were strongly upregulated. To infer about the toxicity of L-homoserine, a transcriptomic analysis of wild-type MG1655 in the presence of 10 mM L-homoserine was performed, which identified a potent repression of locomotion-motility-chemotaxis process. Since the magnitude of this repression was reduced in a ΔthrL mutant concomitantly with a twofold lower sensitivity of this mutant to L-homoserine, one could argue that the repression of this biological process contributed to growth inhibition by L-homoserine. Furthermore, in both wild type and thrl mutant, a strong repression of the branched-chain amino acids synthesis and transport, as well as activation of the sulphate assimilation pathway process to cysteine synthesis were observed in the presence of L-homoserine, which may also contribute to toxic effect of this compound. How this non-canonical amino acid triggers these transcriptomic changes is discussed. Overall design: Transcriptomic analysis implicated 4 strains, namely MG1655 (WT), strain deleted of thrl (Dthrl), strain bearing thrL* allele (thrL*) and strain obtained after adapted laboratory evolution on homoserine (4E). Agilent microarray platfprm GPL13359 was used for microarray analysis . The four strains were inoculated at 0D600 of 0.2 in M9 minimal medium containing 10 g/L glucose in the absence or presence of 10 mM L-homoserine. When cultures reached OD600 of around 1.0, samples were taken for RNA was extraction. Notice that strain 4E is referred in the transcriptomic samples as E4

INSTRUMENT(S): Agilent-020097 E. coli Gene Expression Microarray (Probe Name version)

SUBMITTER: JEAN MARIE FRANCOIS  

PROVIDER: GSE206196 | GEO | 2022-06-18

REPOSITORIES: GEO

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