Engineering Acinetobacter baylyi ADP1 for mevalonate production from lignin-derived aromatic compounds.
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ABSTRACT:
SUBMITTER: Arvay E
PROVIDER: S-EPMC8367835 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Microbial production of intracellular compounds can be engineered by 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 first 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. Second, 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.
| S-EPMC9618324 | biostudies-literature
Project description:Acinetobacter baylyi ADP1 is naturally competent and proficient at homologous recombination, so it can be transformed without restriction digests or ligation reactions. Expression vectors for this system, however, are not yet widely available. Here we describe the construction and characterization of inducible expression vectors that replicate as plasmids in A. baylyi or integrate into a nonessential part of its chromosome. These tools will facilitate the engineering of genes and genomes in this promising model organism.
| S-EPMC3255645 | biostudies-literature
Project description:One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1's native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.
| S-EPMC7192602 | biostudies-literature
Project description:Metabolic engineering can be used as a powerful tool to redirect cell resources towards product synthesis, also in conditions that are not optimal for the production. An example of synthesis strongly dependent on external conditions is the production of storage lipids, which typically requires a high carbon/nitrogen ratio. This requirement also limits the use of abundant nitrogen-rich materials, such as industrial protein by-products, as substrates for lipid production. Acinetobacter baylyi ADP1 is known for its ability to produce industrially interesting storage lipids, namely wax esters (WEs). Here, we engineered A. baylyi ADP1 by deleting the gene aceA encoding for isocitrate lyase and overexpressing fatty acyl-CoA reductase Acr1 in the wax ester production pathway to allow redirection of carbon towards WEs. This strategy led to 3-fold improvement in yield (0.075 g/g glucose) and 3.15-fold improvement in titer (1.82 g/L) and productivity (0.038 g/L/h) by a simple one-stage batch cultivation with glucose as carbon source. The engineered strain accumulated up to 27% WEs of cell dry weight. The titer and cellular WE content are the highest reported to date among microbes. We further showed that the engineering strategy alleviated the inherent requirement for high carbon/nitrogen ratio and demonstrated the production of wax esters using nitrogen-rich substrates including casamino acids, yeast extract, and baker's yeast hydrolysate, which support biomass production but not WE production in wild-type cells. The study demonstrates the power of metabolic engineering in overcoming natural limitations in the production of storage lipids.
| S-EPMC7251950 | biostudies-literature
Project description: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.
| S-EPMC8788691 | biostudies-literature
Project description:Bacterial contact-dependent growth inhibition (CDI) systems are two-partner secretion systems in which toxic CdiA proteins are exported on the outer membrane by cognate transporter CdiB proteins. Upon binding to specific receptors, the C-terminal toxic (CT) domain, detached from CdiA, is delivered to neighbouring cells. Contacts inhibit the growth of not-self-bacteria, lacking immunity proteins co-expressed with CdiA, but promote cooperative behaviours in "self" bacteria, favouring the formation of biofilm structures. The Acinetobacter baylyi ADP1 strain features two CdiA, which differ significantly in size and have different CT domains. Homologous proteins sharing the same CT domains have been identified in A. baumannii. The growth inhibition property of the two A. baylyi CdiA proteins was supported by competition assays between wild-type cells and mutants lacking immunity genes. However, neither protein plays a role in biofilm formation or adherence to epithelial cells, as proved by assays carried out with knockout mutants. Inhibitory and stimulatory properties may be similarly uncoupled in A. baumannii proteins.
| S-EPMC6182759 | biostudies-literature
Project description:Horizontal Gene Transfer, Fitness Costs and Mobility shape the spread of antibiotic resistance genes from Acinetobacter baumannii into experimental populations of Acinetobacter baylyi.
| PRJNA865004 | ENA
Project description:BACKGROUND:Acinetobacter baylyi ADP1 is an ideal bacterial strain for high-throughput genetic analysis as the bacterium is naturally transformable. Thus, ADP1 can be used to investigate DNA mismatch repair, a mechanism for repairing mismatched bases. We used the mutS deletion mutant (XH439) and mutL deletion mutant (XH440), and constructed a mutS mutL double deletion mutant (XH441) to investigate the role of the mismatch repair system in A. baylyi. RESULTS:We determined the survival rates after UV irradiation and measured the mutation frequencies, rates and spectra of wild-type ADP1 and mutSL mutant via rifampin resistance assay (RifR assay) and experimental evolution. In addition, transformation efficiencies of genomic DNA in ADP1 and its three mutants were determined. Lastly, the relative growth rates of the wild type strain, three constructed deletion mutants, as well as the rifampin resistant mutants obtained from RifR assays, were measured. All three mutants had higher survival rates after UV irradiation than wild type, especially the double deletion mutant. Three mutants showed higher mutation frequencies than ADP1 and favored transition mutations in RifR assay. All three mutants showed increased mutation rates in the experimental evolution. However, only XH439 and XH441 had higher mutation rates than the wild type strain in RifR assay. XH441 showed higher transformation efficiency than XH438 when donor DNA harbored transition mutations. All three mutants showed higher growth rates than wild-type, and these four strains displayed higher growth rates than almost all their rpoB mutants. The growth rate results showed different amino acid mutations in rpoB resulted in different extents of reduction in the fitness of rifampin resistant mutants. However, the fitness cost brought by the same mutation did not vary with strain background. CONCLUSIONS:We demonstrated that inactivation of both mutS and mutL increased the mutation rates and frequencies in A. baylyi, which would contribute to the evolution and acquirement of rifampicin resistance. The mutS deletion is also implicated in increased mutation rates and frequencies, suggesting that MutL may be activated even in the absence of mutS. The correlation between fitness cost and rifampin resistance mutations in A. baylyi is firstly established.
| S-EPMC7048072 | biostudies-literature
Project description:Recombination between insertion sequence copies can cause genetic deletion, inversion, or duplication. However, it is difficult to assess the fraction of all genomic rearrangements that involve insertion sequences. In previous gene duplication and amplification studies of Acinetobacter baylyi ADP1, an insertion sequence was evident in approximately 2% of the characterized duplication sites. Gene amplification occurs frequently in all organisms and has a significant impact on evolution, adaptation, drug resistance, cancer, and various disorders. To understand the molecular details of this important process, a previously developed system was used to analyze gene amplification in selected mutants. The current study focused on amplification events in two chromosomal regions that are near one of six copies of the only transposable element in ADP1, IS1236 (an IS3 family member). Twenty-one independent mutants were analyzed, and in contrast to previous studies of a different chromosomal region, IS1236 was involved in 86% of these events. IS1236-mediated amplification could occur through homologous recombination between insertion sequences on both sides of a duplicated region. However, this mechanism presupposes that transposition generates an appropriately positioned additional copy of IS1236. To evaluate this possibility, PCR and Southern hybridization were used to determine the chromosomal configurations of amplification mutants involving IS1236. Surprisingly, the genomic patterns were inconsistent with the hypothesis that intramolecular homologous recombination occurred between insertion sequences following an initial transposition event. These results raise a novel possibility that the gene amplification events near the IS1236 elements arise from illegitimate recombination involving transposase-mediated DNA cleavage.
| S-EPMC3416208 | biostudies-literature
Project description:Genotypic and phenotypic analyses were carried out to clarify the taxonomic position of the naturally transformable Acinetobacter sp. strain ADP1. Transfer tDNA-PCR fingerprinting, 16S rRNA gene sequence analysis, and selective restriction fragment amplification (amplified fragment length polymorphism analysis) indicate that strain ADP1 and a second transformable strain, designated 93A2, are members of the newly described species Acinetobacter baylyi. Transformation assays demonstrate that the A. baylyi type strain B2(T) and two other originally identified members of the species (C5 and A7) also have the ability to undergo natural transformation at high frequencies, confirming that these five strains belong to a separate species of the genus Acinetobacter, characterized by the high transformability of its strains that have been cultured thus far.
| S-EPMC1352221 | biostudies-literature