Development of a genetic toolset for the highly engineerable and metabolically versatile <i>Acinetobacter baylyi</i> 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:10.1101/696302

Cited by 6 publications

(6 citation statements)

References 44 publications

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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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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

Self Cite

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“…This might present an opportunity for the engineering biology approach. Indeed the complexity of the tasks to engineer all the different functions into a single microbial biocatalyst could have been tailor-made for the iteration of the DBTL cycle in the biofoundry [78].…”

Section: Distributed Manufacturingmentioning

confidence: 99%

Addressing the post‐COVID era through engineering biology

Bell

Philp

Kitney

2021

Engineering Biology

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Currently, the world is faced with two fundamental issues of great importance, namely climate change and the coronavirus pandemic. These are intimately involved with the need to control climate change and the need to switch from high carbon, unsustainable economies to low carbon economies. Inherent in this approach are the concepts of the bioeconomy and the Green Industrial Revolution. The article addresses both issues, but it, principally, focusses on the development of the bioeconomy. It considers how nations are divided in relation to the use of biotechnology and synthetic biology in terms of their bioeconomy strategies. The article addresses, as a central theme, the nature and role of engineering biology in these developments. Engineering biology is addressed in terms of BioDesign, coupled with high levels of automation (including AI and machine learning) to increase reproducibility and reliability to meet industrial standards. This lends itself to distributed manufacturing of products in a range of fields. Engineering biology is a platform technology that can be applied in a range of sectors. The bioeconomy, as an engine for economic growth is addressed—in terms of moving from oil‐based economies to bio‐based economies—using biomass, for example, using selected lignocellulosic waste as a feedstock.

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“…ADP1 represents an emerging model organism for research and engineering (Metzgar, 2004; Cuff et al ., 2012; Tumen-Velasquez et al ., 2018; Biggs et al ., 2020; Santala and Santala, 2021). It does not only share most of the traits that make E. coli a convenient model organism but also possesses unique features, such as natural competence and genetic tractability (Metzgar, 2004).…”

Section: Introductionmentioning

confidence: 99%

“…ADP1 represents an emerging model organism for research and engineering (Metzgar, 2004;Cuff et al, 2012;Tumen-Velasquez et al, 2018;Biggs et al, 2020;.…”

Section: Introductionmentioning

confidence: 99%

Engineering cell morphology by CRISPR interference in Acinetobacter baylyi ADP1

Luo

Efimova

Volke

et al. 2022

Preprint

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Microbial production of intracellular compounds can be engineered by, for example, redirecting the carbon flux towards products and increasing the cell size. Potential engineering strategies include exploiting clustered regularly interspaced short palindromic repeats interference (CRISPRi)-based tools for controlling gene expression. Here, we applied CRISPRi for engineering Acinetobacter baylyi ADP1, a model bacterium for synthesizing intracellular storage lipids, namely wax esters. We firstly established an inducible CRISPRi system for strain ADP1, which enables tightly controlled repression of target genes. We then targeted the glyoxylate shunt to redirect carbon flow towards wax esters. Secondly, we successfully employed CRISPRi for modifying cell morphology by repressing ftsZ, an essential gene required for cell division, in combination with targeted knock-outs to generate significantly enlarged filamentous or spherical cells, respectively. The engineered cells sustained increased wax ester production metrics, demonstrating the potential of cell morphology engineering in the production of intracellular lipids.

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

Cited by 6 publications

References 44 publications

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

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

Addressing the post‐COVID era through engineering biology

Engineering cell morphology by CRISPR interference in Acinetobacter baylyi ADP1

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