Vibrio natriegens, the fastest growing non-pathogenic microorganism known to date, has emerged as a highly promising chassis strain for synthetic biology and biotechnology applications. This study analysed the make-up and regulation of the biosynthetic pathway for L-lysine and related L-aspartate family amino acids (AFAAs) in V. natriegens DSM759 to provide a comprehensive basis for future metabolic engineering endeavours aiming at developing this strain into an amino acid overproducer. The compilation of automatically annotated genome sequencing data revealed the presence of gene duplicates encoding putative isozymes for multiple enzymatic reactions within these pathways. The physiological role of these isozymes was analysed via growth phenotyping of corresponding gene deletion mutants as well as enzymatic assays. We verified the presence of a previously unknown mono-functional aspartate kinase isozyme, here termed Vn.LysC2, which was shown to be insensitive to allosteric inhibition by any AFAA. In addition, functional duplicates of L-aspartate semialdehyde dehydrogenase and dihydrodipicolinate synthase enzymes were identified. RNA sequencing experiments were used to elucidate the transcriptional regulation mediated by AFAAs on both their corresponding biosynthetic pathways as well as on the global metabolism. The presence of L-lysine, L-threonine, L-isoleucine and L-methionine resulted in the transcriptional repression of their respective biosynthetic pathways. A global analysis of the transcriptional response revealed that the transcriptional response to L-lysine and L-isoleucine was characterised by a high degree of specificity (four and seven differentially expressed genes, respectively), while L-methionine and L-threonine supplementation affected the expression of a larger number of genes (37 and 60 differentially expressed genes).
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