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The regulated uptake and consumption of d-amino acids by bacteria remain largely unexplored, despite the physiological importance of these compounds. Unlike other characterized bacteria, such as Escherichia coli, which utilizes only l-Asp, Acinetobacter baylyi ADP1 can consume both d-Asp and l-Asp as the sole carbon or nitrogen source. As described here, two LysR-type transcriptional regulators (LTTRs), DarR and AalR, control d- and l-Asp metabolism in strain ADP1. Heterologous expression of A. baylyi proteins enabled E. coli to use d-Asp as the carbon source when either of two transporters (AspT or AspY) and a racemase (RacD) were coexpressed. A third transporter, designated AspS, was also discovered to transport Asp in ADP1. DarR and/or AalR controlled the transcription of aspT, aspY, racD, and aspA (which encodes aspartate ammonia lyase). Conserved residues in the N-terminal DNA-binding domains of both regulators likely enable them to recognize the same DNA consensus sequence (ATGC-N(7)-GCAT) in several operator-promoter regions. In strains lacking AalR, suppressor mutations revealed a role for the ClpAP protease in Asp metabolism. In the absence of the ClpA component of this protease, DarR can compensate for the loss of AalR. ADP1 consumed l- and d-Asn and l-Glu, but not d-Glu, as the sole carbon or nitrogen source using interrelated pathways. IMPORTANCE A regulatory scheme was revealed in which AalR responds to l-Asp and DarR responds to d-Asp, a molecule with critical signaling functions in many organisms. The RacD-mediated interconversion of these isomers causes overlap in transcriptional control in A. baylyi. Our studies improve understanding of transport and regulation and lay the foundation for determining how regulators distinguish l- and d-enantiomers. These studies are relevant for biotechnology applications, and they highlight the importance of d-amino acids as natural bacterial growth substrates.

Adaptive laboratory evolution (ALE) is a powerful approach for improving phenotypes of microbial hosts. Evolved strains typically contain numerous mutations that can be revealed by whole-genome sequencing. However, determining the contribution of specific mutations to new phenotypes is typically challenging and laborious. This task is complicated by factors such as the mutation type, the genomic context, and the interplay between different mutations. Here, a novel approach was developed to identify the significance of mutations in strains evolved from Acinetobacter baylyi ADP1. This method, termed rapid advantageous mutation screening and selection (RAMSES), was used to analyze mutants that emerged from stepwise adaptation to and consumption of high levels of ferulate, a common lignin-derived aromatic compound. After whole-genome sequence analysis, RAMSES allowed rapid determination of effective mutations and seamless introduction of the beneficial mutations into the chromosomes of new strains with different genetic backgrounds. This simple approach to reverse engineering exploits the natural competence and high recombination efficiency of ADP1. Mutated DNA, added directly to growing cells, replaces homologous chromosomal regions to generate transformants that will become enriched if there is a selective benefit. Thus, advantageous mutations can be rapidly identified. Here, the growth advantage of transformants under selective pressure revealed key mutations in genes related to aromatic transport, including hcaE, hcaK, and vanK, and a gene, ACIAD0482, which is associated with lipopolysaccharide synthesis. This study provided insights into the enhanced utilization of industrially relevant aromatic substrates and demonstrated the use of A. baylyi ADP1 as a convenient platform for strain development and evolution studies. IMPORTANCE Microbial conversion of lignin-enriched streams is a promising approach for lignin valorization. However, the lignin-derived aromatic compounds are toxic to cells at relevant concentrations. Although adaptive laboratory evolution (ALE) is a powerful approach to develop more tolerant strains, it is typically laborious to identify the mechanisms underlying phenotypic improvement. We employed Acinetobacter baylyi ADP1, an aromatic-compound-degrading strain that may be useful for biotechnology. The natural competence and high recombination efficiency of this strain can be exploited for critical applications, such as the breakdown of lignin and plastics and abundant polymers composed of aromatic subunits. The natural transformability of this bacterium enabled us to develop a novel approach for rapid screening of advantageous mutations from ALE-derived, aromatic-tolerant, ADP1-derived strains. We clarified the mechanisms and genetic targets for improved tolerance toward common lignin-derived aromatic compounds. This study facilitates metabolic engineering for lignin valorization.

Adaptive laboratory evolution (ALE) is a powerful approach for improving phenotypes of microbial hosts. Evolved strains typically contain numerous mutations that can be revealed by whole-genome sequencing. However, determining the contribution of specific mutations to new phenotypes is typically challenging and laborious. This task is complicated by factors such as the mutation type, the genomic context, and the interplay between different mutations. Here, a novel approach was developed to identify the significance of mutations in strains derived from Acinetobacter baylyi ADP1. This method, termed Rapid Advantageous Mutation ScrEening and Selection (RAMSES), was used to analyze mutants that emerged from stepwise adaptation to, and consumption of, high levels of ferulate, a common lignin-derived aromatic compound. After whole-genome sequence analysis, RAMSES allowed both rapid determination of effective mutations and seamless introduction of the beneficial mutations into the chromosomes of new strains with different genetic backgrounds. This simple approach to reverse-engineering exploits the natural competence and high recombination efficiency of ADP1. The growth advantage of transformants under selective pressure revealed key mutations in genes related to aromatic transport, including hcaE, hcaK, and vanK, and a gene, ACIAD0482, which is associated with lipopolysaccharide synthesis. This study provides insights into enhanced utilization of industrially relevant aromatic substrates and demonstrates the use of A. baylyi ADP1 as a convenient platform for strain development and evolution studies.ImportanceMicrobial conversion of lignin-enriched streams is a promising approach for lignin valorization. However, the lignin-derived aromatic compounds are toxic to cells at relevant concentrations. Adaptive laboratory evolution is a powerful approach to develop more tolerant strains, but revealing the underlying mechanisms behind phenotypic improvement typically involves laborious processes. We employed Acinetobacter baylyi ADP1, an aromatic compound degrading strain that may be useful for biotechnology. The natural competence and high recombination efficiency of strain ADP1 can be exploited for critical applications such as the breakdown of lignin and plastics, abundant polymers composed of aromatic subunits. The natural transformability of this bacterium enabled us to develop a novel approach that allows rapid screening of advantageous mutations from ALE-derived aromatic-tolerant ADP1 strains. We clarified the mechanisms and genetic targets for improved tolerance towards common lignin-derived aromatic compounds. This study facilitates metabolic engineering for lignin valorization.