Development of a genetic toolset for the highly engineerable and metabolically versatile Acinetobacter baylyi ADP1

Biggs, Bradley W.; Bedore, Stacy R.; Arvay, Erika; Huang, Shao-Liang; Subramanian, Harshith; McIntyre, Emily; Duscent-Maitland, Chantel V.; Neidle, Ellen L.; Tyo, Keith E.J.

doi:10.1093/nar/gkaa167

Cited by 38 publications

(32 citation statements)

References 57 publications

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“…In-frame deletions were confirmed by PCR using primers ~150 bp up- and downstream of the introduced mutation, and fusions were confirmed by sequencing. Complementation strains were constructed by placing the gene of interest under a constitutive P tac promoter at the vanAB locus, which has previously been established as a site for chromosomal expression 33 . First, the vanAB genes were replaced with a kanR cassette as described above.…”

Section: Methodsmentioning

confidence: 99%

Acinetobacter baylyi regulates type IV pilus synthesis by employing two extension motors and a motor protein inhibitor

et al. 2021

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Bacteria use extracellular appendages called type IV pili (T4P) for diverse behaviors including DNA uptake, surface sensing, virulence, protein secretion, and twitching motility. Dynamic extension and retraction of T4P is essential for their function, and T4P extension is thought to occur through the action of a single, highly conserved motor, PilB. Here, we develop Acinetobacter baylyi as a model to study T4P by employing a recently developed pilus labeling method. By contrast to previous studies of other bacterial species, we find that T4P synthesis in A. baylyi is dependent not only on PilB but also on an additional, phylogenetically distinct motor, TfpB. Furthermore, we identify a protein (CpiA) that inhibits T4P extension by specifically binding and inhibiting PilB but not TfpB. These results expand our understanding of T4P regulation and highlight how inhibitors might be exploited to disrupt T4P synthesis.

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Section: Methodsmentioning

confidence: 99%

Acinetobacter baylyi regulates type IV pilus synthesis by employing two extension motors and a motor protein inhibitor

et al. 2021

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“…Salvachúa et al examined fourteen bacteria for their ability to utilize a lignin-enriched substrate (26); A. baylyi ADP1 was the best among the tested bacteria both to degrade high molecular weight lignin and consume low molecular weight lignin-derived compounds. The use of A. baylyi ADP1 as a host of engineering for biological lignin valorization is further warranted by its straightforward genome editing (14,46) and the rapidly increasing number of available genetic tools (46).…”

Section: Discussionmentioning

confidence: 99%

Characterization of highly ferulate-tolerant Acinetobacter baylyi ADP1 isolates by a rapid reverse-engineering method

Luo

McIntyre

Bedore

et al. 2021

Preprint

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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.

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“…What is certain, however, is that very little of the search space has ever been covered by natural evolution. A nice example of this is given by the work of Wu et al [230] who used a transformer model (see [39,[231][232][233][234][235][236][237][238][239][240]) [270] Random variation on a trc promoter, allowing 60-fold variation in expression levels [263] Promoter library module combinatorics for use in threonine production [271] Reviews [215,[272][273][274][275][276][277][278][279]] Sigma-factor-specific promoters [280] Transcription factor engineering See below DNA/RNA polymerase engineering [281][282][283][284] Chromosomal integration site Increased isobutanol production from E. coli, involving chromosomal integration at random sites, selection by cell sorting [285] β-carotene synthesis in Yarrowia lipolytica by simultaneous integration of a 3-module biosynthetic pathway plus selection by colony colour.…”

Section: Transcription Vs Translation Engineeringmentioning

confidence: 99%

“…Recently advances in CRISPR and other molecular biology techniques have allowed the integration of reporter genes into a high number of defined genomic sites. Significant variations in expression levels of reporter proteins by the site of genomic integration have been demonstrated in Saccharomyces [ 258 ], E. coli [ 259 , 260 ], Bacillus subtilis [ 261 ], Pseudomonas putida [ 262 ], and Acinetobacter baylyi [ 263 ]. Generally higher levels of expression are seen for integration at sites closer to the origin of replication, as during replication there is in essence a higher copy number of genes that are on the DNA strands replicated first [ 264 ].…”

Section: Transcription Vs Translation Engineeringmentioning

confidence: 99%

Intelligent host engineering for metabolic flux optimisation in biotechnology

Munro

Kell

2021

Biochemical Journal

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Optimising the function of a protein of length N amino acids by directed evolution involves navigating a ‘search space’ of possible sequences of some 20N. Optimising the expression levels of P proteins that materially affect host performance, each of which might also take 20 (logarithmically spaced) values, implies a similar search space of 20P. In this combinatorial sense, then, the problems of directed protein evolution and of host engineering are broadly equivalent. In practice, however, they have different means for avoiding the inevitable difficulties of implementation. The spare capacity exhibited in metabolic networks implies that host engineering may admit substantial increases in flux to targets of interest. Thus, we rehearse the relevant issues for those wishing to understand and exploit those modern genome-wide host engineering tools and thinking that have been designed and developed to optimise fluxes towards desirable products in biotechnological processes, with a focus on microbial systems. The aim throughput is ‘making such biology predictable’. Strategies have been aimed at both transcription and translation, especially for regulatory processes that can affect multiple targets. However, because there is a limit on how much protein a cell can produce, increasing kcat in selected targets may be a better strategy than increasing protein expression levels for optimal host engineering.

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Development of a genetic toolset for the highly engineerable and metabolically versatile Acinetobacter baylyi ADP1

Cited by 38 publications

References 57 publications

Acinetobacter baylyi regulates type IV pilus synthesis by employing two extension motors and a motor protein inhibitor

Acinetobacter baylyi regulates type IV pilus synthesis by employing two extension motors and a motor protein inhibitor

Characterization of highly ferulate-tolerant Acinetobacter baylyi ADP1 isolates by a rapid reverse-engineering method

Intelligent host engineering for metabolic flux optimisation in biotechnology

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