Effect of chemotherapeutic agents on natural transformation frequency in Acinetobacter baylyi.

Natural transformation is the ability of a bacterial cell to take up extracellular DNA which is subsequently available for recombination into the chromosome (or maintenance as an extrachromosomal element). Like other mechanisms of horizontal gene transfer, natural transformation is a significant driver for the dissemination of antimicrobial resistance. Recent studies have shown that many pharmaceutical compounds such as antidepressants and anti-inflammatory drugs can upregulate transformation frequency in the model species Acinetobacter baylyi. Chemotherapeutic compounds have been shown to increase the abundance of antimicrobial resistance genes and increase colonization rates of potentially pathogenic bacteria in patient gastrointestinal tracts, indicating an increased risk of infection and providing a pool of pathogenicity or resistance genes for transformable commensal bacteria. We here test for the effect of six cancer chemotherapeutic compounds on A. baylyi natural transformation frequency, finding two compounds, docetaxel and daunorubicin, to significantly decrease transformation frequency, and daunorubicin to also decrease growth rate significantly. Enhancing our understanding of the effect of chemotherapeutic compounds on the frequency of natural transformation could aid in preventing the horizontal spread of antimicrobial resistance genes.

Carbon-wise utilization of lignin-related compounds by synergistically employing anaerobic and aerobic bacteria.

BACKGROUND: Lignin is a highly abundant but strongly underutilized natural resource that could serve as a sustainable feedstock for producing chemicals by microbial cell factories. Because of the heterogeneous nature of the lignin feedstocks, the biological upgrading of lignin relying on the metabolic routes of aerobic bacteria is currently considered as the most promising approach. However, the limited substrate range and the inefficient catabolism of the production hosts hinder the upgrading of lignin-related aromatics. Particularly, the aerobic O-demethylation of the methoxyl groups in aromatic substrates is energy-limited, inhibits growth, and results in carbon loss in the form of CO2. RESULTS: In this study, we present a novel approach for carbon-wise utilization of lignin-related aromatics by the integration of anaerobic and aerobic metabolisms. In practice, we employed an acetogenic bacterium Acetobacterium woodii for anaerobic O-demethylation of aromatic compounds, which distinctively differs from the aerobic O-demethylation; in the process, the carbon from the methoxyl groups is fixed together with CO2 to form acetate, while the aromatic ring remains unchanged. These accessible end-metabolites were then utilized by an aerobic bacterium Acinetobacter baylyi ADP1. By utilizing this cocultivation approach, we demonstrated an upgrading of guaiacol, an abundant but inaccessible substrate to most microbes, into a plastic precursor muconate, with a nearly equimolar yields (0.9 mol/mol in a small-scale cultivation and 1.0 mol/mol in a one-pot bioreactor cultivation). The process required only a minor genetic engineering, namely a single gene knock-out. Noticeably, by employing a metabolic integration of the two bacteria, it was possible to produce biomass and muconate by utilizing only CO2 and guaiacol as carbon sources. CONCLUSIONS: By the novel approach, we were able to overcome the issues related to aerobic O-demethylation of methoxylated aromatic substrates and demonstrated carbon-wise conversion of lignin-related aromatics to products with yields unattainable by aerobic processes. This study highlights the power of synergistic integration of distinctive metabolic features of bacteria, thus unlocking new opportunities for harnessing microbial cocultures in upgrading challenging feedstocks.

Enhanced upgrading of lignocellulosic substrates by coculture of Saccharomyces cerevisiae and Acinetobacter baylyi ADP1.

BACKGROUND: Lignocellulosic biomass as feedstock has a huge potential for biochemical production. Still, efficient utilization of hydrolysates derived from lignocellulose is challenged by their complex and heterogeneous composition and the presence of inhibitory compounds, such as furan aldehydes. Using microbial consortia where two specialized microbes complement each other could serve as a potential approach to improve the efficiency of lignocellulosic biomass upgrading. RESULTS: This study describes the simultaneous inhibitor detoxification and production of lactic acid and wax esters from a synthetic lignocellulosic hydrolysate by a defined coculture of engineered Saccharomyces cerevisiae and Acinetobacter baylyi ADP1. A. baylyi ADP1 showed efficient bioconversion of furan aldehydes present in the hydrolysate, namely furfural and 5-hydroxymethylfurfural, and did not compete for substrates with S. cerevisiae, highlighting its potential as a coculture partner. Furthermore, the remaining carbon sources and byproducts of S. cerevisiae were directed to wax ester production by A. baylyi ADP1. The lactic acid productivity of S. cerevisiae was improved approximately 1.5-fold (to 0.41 ± 0.08 g/L/h) in the coculture with A. baylyi ADP1, compared to a monoculture of S. cerevisiae. CONCLUSION: The coculture of yeast and bacterium was shown to improve the consumption of lignocellulosic substrates and the productivity of lactic acid from a synthetic lignocellulosic hydrolysate. The high detoxification capacity and the ability to produce high-value products by A. baylyi ADP1 demonstrates the strain to be a potential candidate for coculture to increase production efficiency and economics of S. cerevisiae fermentations.

The recombination initiation functions DprA and RecFOR suppress microindel mutations in Acinetobacter baylyi ADP1.

Short-Patch Double Illegitimate Recombination (SPDIR) has been recently identified as a rare mutation mechanism. During SPDIR, ectopic DNA single-strands anneal with genomic DNA at microhomologies and get integrated during DNA replication, presumably acting as primers for Okazaki fragments. The resulting microindel mutations are highly variable in size and sequence. In the soil bacterium Acinetobacter baylyi, SPDIR is tightly controlled by genome maintenance functions including RecA. It is thought that RecA scavenges DNA single-strands and renders them unable to anneal. To further elucidate the role of RecA in this process, we investigate the roles of the upstream functions DprA, RecFOR, and RecBCD, all of which load DNA single-strands with RecA. Here we show that all three functions suppress SPDIR mutations in the wildtype to levels below the detection limit. While SPDIR mutations are slightly elevated in the absence of DprA, they are strongly increased in the absence of both DprA and RecA. This SPDIR-avoiding function of DprA is not related to its role in natural transformation. These results suggest a function for DprA in combination with RecA to avoid potentially harmful microindel mutations, and offer an explanation for the ubiquity of dprA in the genomes of naturally non-transformable bacteria.

Regulation of tricarboxylate transport and metabolism in Acinetobacter baylyi ADP1.

Despite the significant presence of plant-derived tricarboxylic acids in some environments, few studies detail the bacterial metabolism of trans-aconitic acid (Taa) and tricarballylic acid (Tcb). In a soil bacterium, Acinetobacter baylyi ADP1, we discovered interrelated pathways for the consumption of Taa and Tcb. An intricate regulatory scheme tightly controls the transport and catabolism of both compounds and may reflect that they can be toxic inhibitors of the tricarboxylic acid cycle. The genes encoding two similar LysR-type transcriptional regulators, TcuR and TclR, were clustered on the chromosome with tcuA and tcuB, genes required for Tcb consumption. The genetic organization differed from that in Salmonella enterica serovar Typhimurium, in which tcuA and tcuB form an operon with a transporter gene, tcuC. In A. baylyi, tcuC was not cotranscribed with tcuAB. Rather, tcuC was cotranscribed with a gene, designated pacI, encoding an isomerase needed for Taa consumption. TcuC appears to transport Tcb and cis-aconitic acid (Caa), the presumed product of PacI-mediated periplasmic isomerization of Taa. Two operons, tcuC-pacI and tcuAB, were transcriptionally controlled by both TcuR and TclR, which have overlapping functions. We investigated the roles of the two regulators in activating transcription of both operons in response to multiple effector compounds, including Taa, Tcb, and Caa.IMPORTANCEIngestion of Taa and Tcb by grazing livestock can cause a serious metabolic disorder called grass tetany. The disorder, which results from Tcb absorption by ruminants, focuses attention on the metabolism of tricarboxylic acids. Additional interest stems from efforts to produce tricarboxylic acids as commodity chemicals. Improved understanding of bacterial enzymes and pathways for tricarboxylic acid metabolism may contribute to new biomanufacturing strategies.

AlmA involved in the long-chain n-alkane degradation pathway in Acinetobacter baylyi ADP1 is a Baeyer-Villiger monooxygenase.

Many Acinetobacter species can grow on n-alkanes of varying lengths (≤C40). AlmA, a unique flavoprotein in these Acinetobacter strains, is the only enzyme proven to be required for the degradation of long-chain (LC) n-alkanes, including C32 and C36 alkanes. Although it is commonly presumed to be a terminal hydroxylase, its role in n-alkane degradation remains elusive. In this study, we conducted physiological, biochemical, and bioinformatics analyses of AlmA to determine its role in n-alkane degradation by Acinetobacter baylyi ADP1. Consistent with previous reports, gene deletion analysis showed that almA was vital for the degradation of LC n-alkanes (C26-C36). Additionally, enzymatic analysis revealed that AlmA catalyzed the conversion of aliphatic 2-ketones (C10-C16) to their corresponding esters, but it did not conduct n-alkane hydroxylation under the same conditions, thus suggesting that AlmA in strain ADP1 possesses Baeyer-Villiger monooxygenase (BVMO) activity. These results were further confirmed by bioinformatics analysis, which revealed that AlmA was closer to functionally identified BVMOs than to hydroxylases. Altogether, the results of our study suggest that LC n-alkane degradation by strain ADP1 possibly follows a novel subterminal oxidation pathway that is distinct from the terminal oxidation pathway followed for short-chain n-alkane degradation. Furthermore, our findings suggest that AlmA catalyzes the third reaction in the LC n-alkane degradation pathway.IMPORTANCEMany microbial studies on n-alkane degradation are focused on the genes involved in short-chain n-alkane (≤C16) degradation; however, reports on the genes involved in long-chain (LC) n-alkane (>C20) degradation are limited. Thus far, only AlmA has been reported to be involved in LC n-alkane degradation by Acinetobacter spp.; however, its role in the n-alkane degradation pathway remains elusive. In this study, we conducted a detailed characterization of AlmA in A. baylyi ADP1 and found that AlmA exhibits Baeyer-Villiger monooxygenase activity, thus indicating the presence of a novel LC n-alkane biodegradation mechanism in strain ADP1.

Testing for the fitness benefits of natural transformation during community-embedded evolution.

Natural transformation is a process where bacteria actively take up DNA from the environment and recombine it into their genome or reconvert it into extra-chromosomal genetic elements. The evolutionary benefits of transformation are still under debate. One main explanation is that foreign allele and gene uptake facilitates natural selection by increasing genetic variation, analogous to meiotic sex. However, previous experimental evolution studies comparing fitness gains of evolved transforming- and isogenic non-transforming strains have yielded mixed support for the 'sex hypothesis.' Previous studies testing the sex hypothesis for natural transformation have largely ignored species interactions, which theory predicts provide conditions favourable to sex. To test for the adaptive benefits of bacterial transformation, the naturally transformable wild-type Acinetobacter baylyi and a transformation-deficient ∆comA mutant were evolved for 5 weeks. To provide strong and potentially fluctuating selection, A. baylyi was embedded in a community of five other bacterial species. DNA from a pool of different Acinetobacter strains was provided as a substrate for transformation. No effect of transformation ability on the fitness of evolved populations was found, with fitness increasing non-significantly in most treatments. Populations showed fitness improvement in their respective environments, with no apparent costs of adaptation to competing species. Despite the absence of fitness effects of transformation, wild-type populations evolved variable transformation frequencies that were slightly greater than their ancestor which potentially could be caused by genetic drift.

Application of magnetic-nanoparticle functionalized whole-cell biosensor array for bioavailability and ecotoxicity estimation at urban contaminated sites.

The bioavailability and ecotoxicity of pollutants are important for urban ecological systems and human health, particularly at contaminated urban sites. Therefore, whole-cell bioreporters are used in many studies to assess the risks of priority chemicals; however, their application is restricted by low throughput for specific compounds and complicated operations for field tests. In this study, an assembly technology for manufacturing Acinetobacter-based biosensor arrays using magnetic nanoparticle functionalization was developed to solve this problem. The bioreporter cells maintained high viability, sensitivity, and specificity in sensing 28 priority chemicals, seven heavy metals, and seven inorganic compounds in a high-throughput manner, and their performance remained acceptable for at least 20 d. We also tested the performance by assessing 22 real environmental soil samples from urban areas in China, and our results showed positive correlations between the biosensor estimation and chemical analysis. Our findings prove the feasibility of the magnetic nanoparticle-functionalized biosensor array to recognize the types and toxicities of multiple contaminants for online environmental monitoring at contaminated sites.

Online detection of alkanes by a biological-phase microextraction and biosensing (BPME-BS) device.

Oil spill incidents occur frequently and threaten ecosystems and human health. Solid-phase microextraction allows direct alkane extraction from environmental matrices to improve the limit of detection but is unable to measure alkanes on site. A biological-phase microextraction and biosensing (BPME-BS) device was developed by immobilising an alkane chemotactic Acinetobacter bioreporter ADPWH_alk in agarose gel to achieve online alkane quantification with the aid of a photomultiplier. The BPME-BS device had a high enrichment factor (average 7.07) and a satisfactory limit of detection (0.075 mg/L) for alkanes. The quantification range was 0.1-100 mg/L, comparable to a gas chromatography flame ionisation detector and better than a bioreporter without immobilisation. ADPWH_alk cells in the BPME-BS device maintained good sensitivity under a wide range of environmental conditions, including pH (4.0-9.0), temperature (20-40 °C), and salinity (0.0-3.0%), and its response remained stable within 30 days at 4 °C. In a 7-day continual measurement, the BPME-BS device successfully visualised the dynamic concentration of alkanes, and a 7-day field test successfully captured an oil spill event, helping in source apportionment and on-scene law enforcement. Our work proved that the BPME-BS device is a powerful tool for online alkane measurement, showing substantial potential for fast detection and rapid response to oil spills on site and in situ.

Overproduction, purification, and transcriptional activity of recombinant Acinetobacter baylyi ADP1 RNA polymerase holoenzyme.

Acinetobacter baylyi is an interesting model organism to investigate bacterial metabolism due to its vast repertoire of metabolic enzymes and ease of genetic manipulation. However, the study of gene expression in vitro is dependent on the availability of its RNA polymerase (RNAp), an essential enzyme in transcription. In this work, we developed a convenient method of producing the recombinant A. baylyi ADP1 RNA polymerase holoenzyme (RNApholo) in E. coli that yields 22 mg of a >96% purity protein from a 1-liter shake flask culture. We further characterized the A. baylyi ADP1 RNApholo kinetic profile using T7 Phage DNA as template and demonstrated that it is a highly transcriptionally active enzyme with an elongation rate of 24 nt/s and a termination efficiency of 94%. Moreover, the A. baylyi ADP1 RNApholo has a substantial sequence identity (∼95%) with the RNApholo from the human pathogen Acinetobacter baumannii. This protein can serve as a source of material for structural and biological studies towards advancing our understanding of genome expression and regulation in Acinetobacter species.

Regulation of l- and d-Aspartate Transport and Metabolism in Acinetobacter baylyi ADP1.

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-N7-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.

Batch Experiments Demonstrating a Two-Stage Bacterial Process Coupling Methanotrophic and Heterotrophic Bacteria for 1-Alkene Production From Methane.

Methane (CH4) is a sustainable carbon feedstock for value-added chemical production in aerobic CH4-oxidizing bacteria (methanotrophs). Under substrate-limited (e.g., oxygen and nitrogen) conditions, CH4 oxidation results in the production of various short-chain organic acids and platform chemicals. These CH4-derived products could be broadened by utilizing them as feedstocks for heterotrophic bacteria. As a proof of concept, a two-stage system for CH4 abatement and 1-alkene production was developed in this study. Type I and Type II methanotrophs, Methylobacter tundripaludum SV96 and Methylocystis rosea SV97, respectively, were investigated in batch tests under different CH4 and air supplementation schemes. CH4 oxidation under either microaerobic or aerobic conditions induced the production of formate, acetate, succinate, and malate in M. tundripaludum SV96, accounting for 4.8-7.0% of consumed carbon from CH4 (C-CH4), while M. rosea SV97 produced the same compounds except for malate, and with lower efficiency than M. tundripaludum SV96, accounting for 0.7-1.8% of consumed C-CH4. For the first time, this study demonstrated the use of organic acid-rich spent media of methanotrophs cultivating engineered Acinetobacter baylyi ADP1 'tesA-undA cells for 1-alkene production. The highest yield of 1-undecene was obtained from the spent medium of M. tundripaludum SV96 at 68.9 ± 11.6 µmol mol Csubstrate -1. However, further large-scale studies on fermenters and their optimization are required to increase the production yields of organic acids in methanotrophs.

Biocides used as material preservatives modify rates of de novo mutation and horizontal gene transfer in bacteria.

Antimicrobial resistance (AMR) is a global health problem with the environment being an important compartment for the evolution and transmission of AMR. Previous studies showed that de-novo mutagenesis and horizontal gene transfer (HGT) by conjugation or transformation - important processes underlying resistance evolution and spread - are affected by antibiotics, metals and pesticides. However, natural microbial communities are also frequently exposed to biocides used as material preservatives, but it is unknown if these substances induce mutagenesis and HGT. Here, we show that active substances used in material preservatives can increase rates of mutation and conjugation in a species- and substance-dependent manner, while rates of transformation are not increased. The bisbiguanide chlorhexidine digluconate, the quaternary ammonium compound didecyldimethylammonium chloride, the metal copper, the pyrethroid-insecticide permethrin, and the azole-fungicide propiconazole increase mutation rates in Escherichia coli, whereas no increases were identified for Bacillus subtilis and Acinetobacter baylyi. Benzalkonium chloride, chlorhexidine and permethrin increased conjugation in E. coli. Moreover, our results show a connection between the RpoS-mediated general stress and the RecA-linked SOS response with increased rates of mutation and conjugation, but not for all biocides. Taken together, our data show the importance of assessing the contribution of material preservatives on AMR evolution and spread.

Periplasmic dehydroshikimate dehydratase combined with quinate oxidation in Gluconobacter oxydans for protocatechuate production.

Protocatechuate (3,4-dihydroxybenzoate) has antioxidant properties and is a raw material for the production of muconic acid, which is a key compound in the synthesis of polymers such as nylon and polyethylene terephthalate. Gluconobacter oxydans strain NBRC3244 has a periplasmic system for oxidation of quinate to produce 3-dehydroquinate. Previously, a periplasmic 3-dehydroshikimate production system was constructed by heterologously expressing Gluconacetobacter diazotrophicus dehydroquinate dehydratase in the periplasm of G. oxydans strain NBRC3244. 3-Dehydroshikimate is converted to protocatechuate by dehydration. In this study, we constructed a G. oxydans strain that expresses the Acinetobacter baylyi quiC gene, which encodes a dehydroshikimate dehydratase of which the subcellular localization is likely the periplasm. We attempted to produce protocatechuate by co-cultivation of two recombinant G. oxydans strains-one expressing the periplasmically targeted dehydroquinate dehydratase and the other expressing A. baylyi dehydroshikimate dehydratase. The co-cultivation system produced protocatechuate from quinate in a nearly quantitative manner.

Characterization of Highly Ferulate-Tolerant Acinetobacter baylyi ADP1 Isolates by a Rapid Reverse Engineering Method.

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.

Insights of metallic nanoparticles and ions in accelerating the bacterial uptake of antibiotic resistance genes.

The increasing release of nanomaterials has attracted significant concerns for human and environmental health. Similarly, the dissemination of antimicrobial resistance (AMR) is a global health crisis affecting approximately 700,000 people a year. However, a knowledge gap persists between the spread of AMR and nanomaterials. This study aims to fill this gap by investigating whether and how nanomaterials could directly facilitate the dissemination of AMR through horizontal gene transfer. Our results show that commonly-used nanoparticles (NPs) (Ag, CuO and ZnO NPs) and their ion forms (Ag+, Cu2+ and Zn2+) at realistic concentrations within aquatic environments can significantly promote the transformation of extracellular antibiotic resistance genes in Acinetobacter baylyi ADP1 by a factor of 11.0-folds, which is comparable to the effects of antibiotics. The enhanced transformation by Ag NPs/Ag+ and CuO NPs/Cu2+ was primarily associated with the overproduction of reactive oxygen species and cell membrane damage. ZnO NPs/Zn2+ might increase the natural transformation rate by stimulating the stress response and ATP synthesis. All tested NPs/ions resulted in upregulating the competence and SOS response-associated genes. These findings highlight a new concern that nanomaterials can speed up the spread of AMR, which should not be ignored when assessing the holistic risk of nanomaterials.

Engineering Acinetobacter baylyi ADP1 for mevalonate production from lignin-derived aromatic compounds.

Utilization of lignin, an abundant renewable resource, is limited by its heterogenous composition and complex structure. Biological valorization of lignin provides advantages over traditional chemical processing as it occurs at ambient temperature and pressure and does not use harsh chemicals. Furthermore, the ability to biologically funnel heterogenous substrates to products eliminates the need for costly downstream processing and separation of feedstocks. However, lack of relevant metabolic networks and low tolerance to degradation products of lignin limits the application of traditional engineered model organisms. To circumvent this obstacle, we employed Acinetobacter baylyi ADP1, which natively catabolizes lignin-derived aromatic substrates through the ß-ketoadipate pathway, to produce mevalonate from lignin-derived compounds. We enabled expression of the mevalonate pathway in ADP1 and validated activity in the presence of multiple lignin-derived aromatic substrates. Furthermore, by knocking out wax ester synthesis and utilizing fed-batch cultivation, we improved mevalonate titers 7.5-fold to 1014 mg/L (6.8 mM). This work establishes a foundation and provides groundwork for future efforts to engineer improved production of mevalonate and derivatives from lignin-derived aromatics using ADP1.

Acinetobacter baylyi ADP1-naturally competent for synthetic biology.

Acinetobacter baylyi ADP1 is a non-pathogenic soil bacterium known for its metabolic diversity and high natural transformation and recombination efficiency. For these features, A. baylyi ADP1 has been long exploited in studying bacterial genetics and metabolism. The large pool of information generated in the fundamental studies has facilitated the development of a broad range of sophisticated and robust tools for the genome and metabolic engineering of ADP1. This mini-review outlines and describes the recent advances in ADP1 engineering and tool development, exploited in, for example, pathway and enzyme evolution, genome reduction and stabilization, and for the production of native and non-native products in both pure and rationally designed multispecies cultures. The rapidly expanding toolbox together with the unique features of A. baylyi ADP1 provide a strong base for a microbial cell factory excelling in synthetic biology applications where evolution meets rational engineering.

Tn1 transposition in the course of natural transformation enables horizontal antibiotic resistance spread in Acinetobacter baylyi.

Transposons are genetic elements that change their intracellular genomic position by transposition and are spread horizontally between bacteria when located on plasmids. It was recently discovered that transposition from fully heterologous DNA also occurs in the course of natural transformation. Here, we characterize the molecular details and constraints of this process using the replicative transposon Tn1 and the naturally competent bacterium Acinetobacter baylyi. We find that chromosomal insertion of Tn1 by transposition occurs at low but detectable frequencies and preferably around the A. baylyi terminus of replication. We show that Tn1 transposition is facilitated by transient expression of the transposase and resolvase encoded by the donor DNA. RecA protein is essential for the formation of a circular, double-stranded cytoplasmic intermediate from incoming donor DNA, and RecO is beneficial but not essential in this process. Absence of the recipient RecBCD nuclease stabilizes the double-stranded intermediate. Based on these results, we suggest a mechanistic model for transposition during natural transformation.

Automating Cloning by Natural Transformation.

Affordable and automated cloning platforms are essential to many synthetic biology studies. However, the traditional E. coli-based cloning is a major bottleneck as it requires heat shock or electroporation implemented in the robotic workflows. To overcome this problem, we explored bacterial natural transformation for automatic DNA cloning and engineering. Recombinant plasmids are efficiently generated from Gibson or overlap extension PCR (OE-PCR) products by simply adding the DNA into Acinetobacter baylyi ADP1 cultures. No DNA purification, competence induction, or special equipment is required. Up to 10,000 colonies were obtained per microgram of DNA, while the number of false positive colonies was low. We cloned and engineered 21 biosynthetic gene clusters (BGCs) of various types, with length from 1.5 to 19 kb and GC content from 35% to 72%. One of them, a nucleoside BGC, showed antibacterial activity. Furthermore, the method was easily transferred to a low-cost benchtop robot with consistent cloning efficiency. Thus, this automatic natural transformation (ANT) cloning provides an easy, robust, and affordable platform for high throughput DNA engineering.