Strain MG1655 Research Articles

Response and adaptation of the transcriptional heat shock response to pressure.

The molecular mechanisms underlying pressure adaptation remain largely unexplored, despite their significance for understanding biological adaptation and improving sterilization methods in the food and beverage industry. The heat shock response leads to a global stabilization of the proteome. Prior research suggested that the heat shock regulon may exhibit a transcriptional response to high-pressure stress. In this study, we investigated the pressure-dependent heat shock response in E. coli strains using plasmid-borne green fluorescent protein (GFP) promoter fusions and fluorescence fluctuation microscopy. We quantitatively confirm that key heat shock genes-rpoH, rpoE, dnaK, and groEL - are transcriptionally upregulated following pressure shock in both piezosensitive Escherichia coli and a more piezotolerant laboratory-evolved strain, AN62. Our quantitative imaging results provide the first single cell resolution measurements for both the heat shock and pressure shock transcriptional responses, revealing not only the magnitude of the responses, but also the biological variance involved. Moreover, our results demonstrate distinct responses in the pressure-adapted strain. Specifically, PgroEL is upregulated more than PdnaK in AN62, while the reverse is true in the parental strain. Furthermore, unlike in the parental strain, the pressure-induced upregulation of PrpoE is highly stochastic in strain AN62, consistent with a strong feedback mechanism and suggesting that RpoE could act as a pressure sensor. Despite its capacity to grow at pressures up to 62 MPa, the AN62 genome shows minimal mutations, with notable single nucleotide substitutions in genes of the transcriptionally important b subunit of RNA polymerase and the Rho terminator. In particular, the mutation in RNAP is one of a cluster of mutations known to confer rifampicin resistance to E. coli via modification of RNAP pausing and termination efficiency. The observed differences in the pressure and heat shock responses between the parental MG1655 strain and the pressure-adapted strain AN62 could arise in part from functional differences in their RNAP molecules.

Genetically modified E. Coli secreting melanin (E.melanin) activates the astrocytic PSAP-GPR37L1 pathway and mitigates the pathogenesis of Parkinson’s disease

The characteristic neuropathology of Parkinson’s disease (PD) involves the abnormal accumulation of phosphorylated α-synuclein (αSyn), as well as a significant decrease in neuromelanin (NM) levels within dopamine neurons (DaNs). Unlike αSyn aggregates, the relationship between NM levels and PD pathogenesis is not well understood. In this study, we engineered an E. coli MG1655 strain to produce exosomes containing melanin (E.melanin), and investigated its potential neuroprotective effects on DaNs in the context of PD. By employing a combination of cell cultures, biochemical studies, single nuclear RNA sequencing (snRNA seq), and various in vivo validations, we found that administration of E.melanin effectively alleviated DaNs loss and improved motor behavior impairments observed in both pharmacological and transgenic PD mouse models. Mechanistically, snRNA seq data suggested that E.melanin activated the PSAP-GPR37L1 signaling pathway specifically within astrocytes, leading to a reduction in astrocytic engulfment of synapses. Notably, activation of the GPR37L1 receptor using Tx14(A) peptide successfully rescued motor defects as well as protected against DaNs degeneration in mice with PD. Overall, our findings provide novel insights into understanding the molecular mechanisms underlying melanin’s protective effects on DaNs in PD while offering potential strategies for manipulating and treating its pathophysiological progression.

Hybridized and engineered microbe for catalytic generation of peroxynitrite and cancer immunotherapy under sonopiezo initiation.

Living therapeutics is an emerging antitumor modality by living microorganisms capable of selective tropism and effective therapeutics. Nevertheless, primitive microbes could only present limited therapeutic functionalities against tumors. Hybridization of the microbes with multifunctional nanocatalysts is of great significance to achieve enhanced tumor catalytic therapy. In the present work, nitric oxide synthase (NOS)-engineered Escherichia coli strain MG1655 (NOBac) was used to hybridize with the sonopiezocatalytic BaTiO3 nanoparticles (BTO NPs) for efficient tumor-targeted accumulation and antitumor therapy. Under ultrasound irradiation, superoxide anions created by the piezocatalytic reaction of BTO NPs could immediately react with nitric oxide (NO) generated from NOBac to produce highly oxidative peroxynitrite ONOO- species in cascade, resulting in robust tumor piezocatalytic therapeutic efficacy, prompting prominent and sustained antitumoral immunoactivation simultaneously. The present work presents a promising cancer immunotherapy based on the engineered and hybridized microbes for highly selective and sonopiezo-controllable tumor catalytic therapy.

New insight into acid-resistant enzymes from natural mutations of Escherichia coli Nissle 1917

The probiotic Escherichia coli Nissle 1917 (EcN), known for its superior acid resistance (AR), serves as a promising chassis for live therapeutics due to the effective colonization capabilities. However, the enzymatic activity regarding AR in EcN remains poorly understood. First, we investigated the AR systems of EcN by measuring cell growth under acidic stress and exploring the relationship of mutations to their corresponding enzymatic activities. As a result, the catalytic activity of inducible decarboxylases of GadB, AdiA and CadA, responsible for metabolizing glutamate, arginine, and lysine, exhibited an average 2-fold increase in EcN compared to the reference strain MG1655. Furthermore, we discovered that the glutamate-dependent AR2 system in EcN was meticulously regulated by specific regulons such as GadW. This study not only revealed the physiology of EcN under acidic conditions, but also highlighted that the mutated core enzymes in the AR system of EcN exhibit improved activities.

Dual-Plasmid Mini-Tn5 System to Stably Integrate Multicopy of Target Genes in Escherichia coli.

The efficiency of valuable metabolite production by engineered microorganisms underscores the importance of stable and controllable gene expression. While plasmid-based methods offer flexibility, integrating genes into host chromosomes can establish stability without selection pressure. However, achieving site-directed multicopy integration presents challenges, including site selection and stability. We introduced a stable multicopy integration method by using a novel dual-plasmid mini-Tn5 system to insert genes into Escherichia coli's genome. The gene of interest was combined with a removable antibiotic resistance gene. After the selection of bacteria with inserted genes, the antibiotic resistance gene was removed. Optimizations yielded an integration efficiency of approximately 5.5 × 10-3 per recipient cell in a single round. Six rounds of integration resulted in 19 and 5 copies of the egfp gene in the RecA+ strain MG1655 and the RecA- strain XL1-Blue MRF', respectively. Additionally, we integrated a polyhydroxybutyrate (PHB) synthesis gene cluster into E. coli MG1655, yielding an 8-copy integration strain producing more PHB than strains with the cluster on a high-copy plasmid. The method was efficient in generating gene insertions in various E. coli strains, and the inserted genes were stable after extended culture. This stable, high-copy integration tool offers potential for diverse applications in synthetic biology.

Modified Tn7 transposon vectors for controlled chromosomal gene expression.

Complementation remains a foundation for demonstrating molecular Koch's postulates. While this is frequently achieved using plasmids, limitations such as increased gene copy number and the need for antibiotic supplementation to avoid plasmid loss can restrict their use. Chromosomal integration systems using the Tn7 transposon provide an alternative to plasmids for complementation and facilitate the stable insertion of genes at the chromosomal attTn7 site without the need for selection pressure. Here, we enhanced the utility of mini-Tn7 insertion vectors by the addition of inducible (Pcym) and constitutive (PcL and PrpsM) promoters, allowing differential transcriptional control of genes integrated into the chromosome. We validated the utility of these promoters by cloning the gfp gene, encoding green fluorescent protein, downstream of each promoter and integrating a mini-Tn7 construct harboring these elements into the attTn7 site on the chromosome of the Escherichia coli K-12 strain MG1655. The PcL and PrpsM promoters provided equivalent levels of GFP expression and offered flexibility based on the target host strain. Activation of the tightly regulated Pcym promoter with its inducer cumate resulted in tunable expression of GFP in a dose-dependent manner. We further demonstrated the tight control of the Pcym promoter using the toxic impCAB genes, and the expression of which is detrimental to E. coli viability. Together, these modified mini-Tn7 vectors allowing differential control of genes integrated into the chromosome at a conserved site offer an efficient system for complementation where plasmid use is restricted.IMPORTANCEChromosomal integration using mini-Tn7 vectors provides an efficient means to insert genes into the chromosome of many gram-negative bacteria. Insertion occurs at a conserved site and allows for the stable integration of genes in single copy. While this system has multiple benefits for enabling complementation, a cornerstone for fulfilling molecular Koch's postulates, greater flexibility for controlled gene expression would enhance its utility. Here, we have added to the function of mini-Tn7 vectors by the addition of inducible and constitutive promoters and demonstrated their capacity to drive the controlled expression of target genes integrated into the chromosome. In addition to complementation, these modified vectors offer broad application for other approaches including chromosomal tagging, in vivo expression, metabolic engineering, and synthetic biology.

A single rareσ70 variant establishes a unique gene expression pattern in the E. coli pathobiont LF82.

LF82, an adherent-invasive Escherichia coli (AIEC) pathobiont, is associated with Crohn's disease, an inflammatory bowel disease of unknown etiology. Although AIEC phenotypes differ from those of 'commensal' or pathogenic E. coli, work has failed to identify genetic features accounting for these differences. We have investigated a natural, but rare, single nucleotide polymorphism (SNP) in LF82 present within the highly conserved rpoD gene, encodingσ70 [primary sigma factor, RNA polymerase (RNAP)]. We demonstrate thatσ70 D445V results in transcriptomicand phenotypic changes consistent with LF82 phenotypes, including increased antibiotic resistance and biofilm formation and increased capacity for methionine biosynthesis. RNA-seq analyses comparingσ70 V445 versus σ70 D445 identified 24 genes upregulated byσ70 V445 in both LF82 and the laboratory E. coli K-12 strain MG1655. Using in vitro transcription, we demonstrate thatσ70 D445V directly increases transcription from promoters for several of the up-regulated genes and that the presence of a 16 bp spacer and -14 G:C is associated with this increase. The position of D445V within RNAP suggests that it could affect RNAP/spacer interaction. Our work represents the first identification of a distinguishing SNP for this pathobiont and suggests an underrecognized mechanism by which pathobionts and strain variants can emerge.

Enzymatic promiscuity and underground reactions accounted for the capability of Escherichia coli to use the non-natural chemical synthon 2,4-dihydroxybutyric acid as a carbon source for growth

2,4-dihydroxybutyric acid (DHB) and 2-keto-4-hydroxybutyrate (OHB) are non-natural molecules obtained through synthetic pathways from renewable carbon source. As they are structurally similar to lactate and pyruvate respectively, they could possibly interfere with the metabolic network of Escherichia coli. In fact, we showed that DHB can be easily oxidized by the membrane associated L and D-lactate dehydrogenases encoded by lldD, dld and ykgF into OHB, and the latter being cleaved into pyruvate and formaldehyde by several pyruvate-dependent aldolases, with YagE being the most effective. While formaldehyde was readily detoxified into formate, Escherichia coli K12 MG1655 strain failed to grow on DHB despite of the production of pyruvate. To find out the reason for this failure, we constructed a mutant strain whose growth was rendered dependent on DHB and subjected this strain to adaptive evolution. Genome sequencing of the adapted strain revealed an essential role for ygbI encoding a transcriptional repressor of the threonate operon in this DHB-dependent growth. This critical function was attributed to the derepression of ygbN encoding a putative threonate transporter, which was found to exclusively transport the D form of DHB. A subsequent laboratory evolution was carried out with E. coli K12 MG1655 deleted for ΔygbI to adapt for growth on DHB as sole carbon source. Remarkably, only two additional mutations were disclosed in the adapted strain, which were demonstrated by reverse engineering to be necessary and sufficient for robust growth on DHB. One mutation was in nanR encoding the transcription repressor of sialic acid metabolic genes, causing 140-fold increase in expression of nanA encoding N-acetyl neuraminic acid lyase, a pyruvate-dependent aldolase, and the other was in the promoter of dld leading to 14-fold increase in D-lactate dehydrogenase activity on DHB. Taken together, this work illustrates the importance of promiscuous enzymes in underground metabolism and moreover, in the frame of synthetic pathways aiming at producing non-natural products, these underground reactions could potentially penalize yield and title of these bio-based products.

Biological Activity of Hybrid Vaterite-Pectin Microparticles Towards Bacteria E. coli and Human Neutrophils.

Interaction of microbiota with hybrid vaterite-pectin microparticles as an attractive multifunctional vehicle for mucosal delivery should not provoke inflammation. Our purpose was to study the reaction of bacteria E. coli strain Mg1655 and isolate SharL from a patient with Crohn disease on the cultivation with hybrid microparticles and vaterite, and the subsequent activation of neutrophils. Vaterite-pectin microparticles enhanced leakage of ATP from bacteria. For E. coli Mg1655, the concentration of DNA decreased, while intracellular ATP increased. For E. coli SharL, the intracellular ATP decreased with simultaneous growth of DNA. Bacteria and microparticles together did not enhance activation of neutrophils in comparison with the particles per se in the medium without serum and in comparison with bacteria in the medium supplemented with serum; microparticles did not reduce functional activity of neutrophils.

Optimizing Antimicrobial Peptide Design: Integration of Cell-Penetrating Peptides, Amyloidogenic Fragments, and Amino Acid Residue Modifications.

The escalating threat of multidrug-resistant pathogens necessitates innovative approaches to combat infectious diseases. In this study, we examined peptides R23FS*, V31KS*, and R44KS*, which were engineered to include an amyloidogenic fragment sourced from the S1 protein of S. aureus, along with one or two cell-penetrating peptide (CPP) components. We assessed the antimicrobial efficacy of these peptides in a liquid medium against various strains of both Gram-positive bacteria, including S. aureus (209P and 129B strains), MRSA (SA 180 and ATCC 43300 strains), and B. cereus (strain IP 5832), and Gram-negative bacteria such as P. aeruginosa (ATCC 28753 and 2943 strains) and E. coli (MG1655 and K12 strains). Peptides R23FS*, V31KS*, and R44KS* exhibited antimicrobial activity comparable to gentamicin and meropenem against all tested bacteria at concentrations ranging from 24 to 48 μM. The peptides showed a stronger antimicrobial effect against B. cereus. Notably, peptide R44KS* displayed high efficacy compared to peptides R23FS* and V31KS*, particularly evident at lower concentrations, resulting in significant inhibition of bacterial growth. Furthermore, modified peptides V31KS* and R44KS* demonstrated enhanced inhibitory effects on bacterial growth across different strains compared to their unmodified counterparts V31KS and R44KS. These results highlight the potential of integrating cell-penetrating peptides, amyloidogenic fragments, and amino acid residue modifications to advance the innovation in the field of antimicrobial peptides, thereby increasing their effectiveness against a broad spectrum of pathogens.

Biosensor-Based Assessment of Pesticides and Mineral Fertilizers’ Influence on Ecotoxicological Parameters of Soils under Soya, Sunflower and Wheat

Pesticides and fertilizers used in agriculture can negatively affect the soil, increasing its toxicity. In this work, a battery of whole-cell bacterial lux-biosensors based on the E. coli MG1655 strain with various inducible promoters, as well as the natural luminous Vibrio aquamarinus VKPM B-11245 strain, were used to assess the effects of agrochemical soil treatments. The advantages of using biosensors are sensitivity, specificity, low cost of analysis, and the ability to assess the total effect of toxicants on a living cell and the type of their toxic effect. Using the V. aquamarinus VKPM B-11245 strain, the synergistic effect of combined soil treatment with pesticides and mineral fertilizers was shown, which led to an increase in the overall (integral) toxicity of soils higher than that of the individual application of substances. Several probable implementation mechanisms of agrochemical toxic effects have been discovered. DNA damage caused by both SOS response induction and alkylation, oxidative stress due to increased superoxide levels, and damage to cellular proteins and membranes are among them. Thus, the usage of biosensors makes it possible to assess the cumulative effect of various toxicants on living organisms without using expensive chemical analyses.

Acquisition of plasmids from Shiga toxin-producingEscherichia colistrains had low or neutral fitness cost on commensalE. coli.

Although it has been hypothesized that the acquisition of plasmids-especially those bearing virulence factors and antimicrobial resistance genes-increases the energetic burden and reduces the fitness of a bacterium in general, some results have challenged this view, showing little or no effect on fitness after plasmid acquisition, which may lead to change in the view that there are evolutionary barriers for a wide spread of such plasmids among bacteria. Here, to evaluate the fitness impact of plasmid-encoded antibiotic resistance and virulence genes, plasmids from O26:H11, O111:H8, and O118:H16 Shiga toxin-producing Escherichia coli (STEC) human and bovine isolates were transferred to the non-virulent E. coli HS and K-12 MG1655 strains. Sequencing and PCR were used to characterize plasmids, and to identify the presence of antimicrobial resistance and/or virulence genes. The fitness impact of plasmids encoding virulence and antimicrobial resistance upon bacterial hosts was determined by pairwise growth competition. Plasmid profile analysis showed that STEC strains carried one or more high and low molecular weight plasmids belonging to the B/O, F, I, K, P, Q, and/or X incompatibility groups encoding virulence genes (SPATE-encodinggenes) and/or antimicrobial resistance genes (aadA1, strAB, tetA, and/or tetB). Competition experiments demonstrated that the biological cost of carriage of these plasmids by the commensal E. coli strain HS or the laboratory strain E. coli K-12 MG1655 was low or non-existent, ranging from - 4.7 to 5.2% per generation. This suggests that there are few biological barriers-or, alternatively, it suggests that there are biological barriers that we were not able to measure in this competition model-against the spread of plasmid encoding virulence and resistance genes from STEC to other, less pathogenic E. coli strains. Thus, our results, in opposition to a common view, suggest that the acquisition of plasmids does not significantly affect the bacteria fitness and, therefore, the theorized plasmid burden would not be a significant barrier for plasmid spread.

Sodium Sulfite as a Novel Hypoxia Revulsant Involved in Hypoxic Regulation in Escherichia coli

As a reducing salt, sodium sulfite could deprive oxygen in solution, which could mimic hypoxic stress in Caenorhabditis elegans. In this study, the wild-type Escherichia coli strain MG1655 was used to examine the inhibition of sodium sulfite-induced hypoxia by observing the bacterial growth curves. We also analyzed the growth curves of mutant strains (for arcA/B, soxR/S, fnr, and oxyR) related to E. coli hypoxic pathways to reveal roles of the related genes during hypoxia. The ultrastructure of hypoxia-inhibited bacteria were also observed using transmission electron microscopy. Sodium sulfite could maintain hypoxic condition of bacterial culture for 8 h with concentrations over 40 mmol/L. Complete ultrastructure of the bacteria indicated sodium sulfite did inhibit bacterial growth and division. Among the hypoxia genes, fnr and arcB played key roles in sodium sulfite-induced hypoxia. This study showed that sodium sulfite could be used as a novel hypoxia revulsant for bacterial cultures.

Genome-wide identification of fitness-genes in aminoglycoside-resistant Escherichia coli during antibiotic stress

Resistance against aminoglycosides is widespread in bacteria. This study aimed to identify genes that are important for growth of E. coli during aminoglycoside exposure, since such genes may be targeted to re-sensitize resistant E. coli to treatment. We constructed three transposon mutant libraries each containing > 230.000 mutants in E. coli MG1655 strains harboring streptomycin (aph(3″)-Ib/aph(6)-Id), gentamicin (aac(3)-IV), or neomycin (aph(3″)-Ia) resistance gene(s). Transposon Directed Insertion-site Sequencing (TraDIS), a combination of transposon mutagenesis and high-throughput sequencing, identified 56 genes which were deemed important for growth during streptomycin, 39 during gentamicin and 32 during neomycin exposure. Most of these fitness-genes were membrane-located (n = 55) and involved in either cell division, ATP-synthesis or stress response in the streptomycin and gentamicin exposed libraries, and enterobacterial common antigen biosynthesis or magnesium sensing/transport in the neomycin exposed library. For validation, eight selected fitness-genes/gene-clusters were deleted (minCDE, hflCK, clsA and cpxR associated with streptomycin and gentamicin resistance, and phoPQ, wecA, lpp and pal associated with neomycin resistance), and all mutants were shown to be growth attenuated upon exposure to the corresponding antibiotics. In summary, we identified genes that are advantageous in aminoglycoside-resistant E. coli during antibiotic stress. In addition, we increased the understanding of how aminoglycoside-resistant E. coli respond to antibiotic exposure.

Zeta potential characterization using commercial microfluidic chips.

Surface charge is a critical feature of microbes that affects their interactions with other cells and their environment. Because bacterial surface charge is difficult to measure directly, it is typically indirectly inferred through zeta potential measurements. Existing tools to perform such characterization are either costly and ill-suited for non-spherical samples or rely on microfluidic techniques requiring expensive fabrication equipment or specialized facilities. Here, we report the application of commercially available PMMA microfluidic chips and open-source data analysis workflows for facile electrokinetic characterization of particles and cells after prior zeta potential measurement with a Zetasizer for calibration. Our workflows eliminate the need for microchannel fabrication, increase measurement reproducibility, and make zeta potential measurements more accessible. This novel methodology was tested with functionalized 1 μm and 2 μm polystyrene beads as well as Escherichia coli MG1655 strain. Measured zeta potentials for these samples were in agreement with literature values obtained by conventional measurement methods. Taken together, our data demonstrate the power of this workflow to broadly enable critical measurements of particle and bacterial zeta potential for numerous applications.

Identifying Key In Silico Knockout for Enhancement of Limonene Yield Through Dynamic Metabolic Modelling.

Living cells display dynamic and complex behaviors. To understand their response and to infer novel insights not possible with traditional reductionist approaches, over the last few decades various computational modelling methodologies have been developed. In this chapter, we focus on modelling the dynamic metabolic response, using linear and nonlinear ordinary differential equations, of an engineered Escherichia coli MG1655 strain with plasmid pJBEI-6409 that produces limonene. We show the systems biology steps involved from collecting time-series data of living cells, to dynamic model creation and fitting the model with experimental responses using COPASI software.

Engineering E. coli strains using antibiotic-resistance-gene-free plasmids

We created a generalizable pipeline for antibiotic-resistance-gene-free plasmid (ARGFP)-based cloning using a dual auxotrophic- and essential-gene-based selection strategy. We use auxotrophic selection to construct plasmids in engineered E.coli DH10B cloning strains and both auxotrophic- and essential-gene-based selection to (1) select for recombinant strains and (2) maintain a plasmid in E.coli Nissle 1917, a common chassis for engineered probiotic applications, and E.coli MG1655, the laboratory "wild-type" E.coli strain. We show that our approach has comparable efficiency to that of antibiotic-resistance-gene-based cloning. We also show that the double-knockout Nissle and MG1655 strains are simple to transform with plasmids of interest. Notably, we show that the engineered Nissle strains are amenable to long-term plasmid maintenance in repeated culturing as well as in the mouse gut, demonstrating the potential for broad applications while minimizing the risk of antibiotic resistance spread via horizontal gene transfer.

O antigen biogenesis sensitises Escherichia coli K-12 to bile salts, providing a plausible explanation for its evolutionary loss.

Escherichia coli K-12 is a model organism for bacteriology and has served as a workhorse for molecular biology and biochemistry for over a century since its first isolation in 1922. However, Escherichia coli K-12 strains are phenotypically devoid of an O antigen (OAg) since early reports in the scientific literature. Recent studies have reported the presence of independent mutations that abolish OAg repeating-unit (RU) biogenesis in E. coli K-12 strains from the same original source, suggesting unknown evolutionary forces have selected for inactivation of OAg biogenesis during the early propagation of K-12. Here, we show for the first time that restoration of OAg in E. coli K-12 strain MG1655 synergistically sensitises bacteria to vancomycin with bile salts (VBS). Suppressor mutants surviving lethal doses of VBS primarily contained disruptions in OAg biogenesis. We present data supporting a model where the transient presence and accumulation of lipid-linked OAg intermediates in the periplasmic leaflet of the inner membrane interfere with peptidoglycan sacculus biosynthesis, causing growth defects that are synergistically enhanced by bile salts. Lastly, we demonstrate that continuous bile salt exposure of OAg-producing MG1655 in the laboratory, can recreate a scenario where OAg disruption is selected for as an evolutionary fitness benefit. Our work thus provides a plausible explanation for the long-held mystery of the selective pressure that may have led to the loss of OAg biogenesis in E. coli K-12; this opens new avenues for exploring long-standing questions on the intricate network coordinating the synthesis of different cell envelope components in Gram-negative bacteria.

Genome engineering allows selective conversions of terephthalaldehyde to multiple valorized products in bacterial cells

AbstractDeconstruction of polyethylene terephthalate (PET) plastic waste generates opportunities for valorization to alternative products. We recently designed an enzymatic cascade that could produce terephthalaldehyde (TPAL) from terephthalic acid. Here, we showed that the addition of TPAL to growing cultures of Escherichia coli wild‐type strain MG1655 and an engineered strain for reduced aromatic aldehyde reduction (RARE) strain resulted in substantial reduction. We then investigated if we could mitigate this reduction using multiplex automatable genome engineering (MAGE) to create an E. coli strain with 10 additional knockouts in RARE. Encouragingly, we found this newly engineered strain enabled a 2.5‐fold higher retention of TPAL over RARE after 24 h. We applied this new strain for the production of para‐xylylenediamine (pXYL) and observed a 6.8‐fold increase in pXYL titer compared with RARE. Overall, our study demonstrates the potential of TPAL as a versatile intermediate in microbial biosynthesis of chemicals that derived from waste PET.

Toxicity of per- and polyfluoroalkyl substances to microorganisms in confined hydrogel structures

Per- and polyfluoroalkyl substances (PFAS) as a group of environmentally persistent synthetic chemicals has been widely used in industrial and consumer products. Bioaccumulation studies have documented the adverse effects of PFAS in various living organisms. Despite the large number of studies, experimental approaches to evaluate the toxicity of PFAS on bacteria in a biofilm-like niche as structured microbial communities are sparse. This study suggests a facile approach to query the toxicity of PFOS and PFOA on bacteria (Escherichia coli K12 MG1655 strain) in a biofilm-like niche provided by hydrogel-based core-shell beads. Our study shows that E. coli MG1655 upon complete confinement in hydrogel beads exhibit altered physiological characteristics of viability, biomass, and protein expression, compared to their susceptible counterpart cultivated under planktonic conditions. We find that soft-hydrogel engineering platforms may provide a protective role for microorganisms from environmental contaminants, depending on the size or thickness of the protective/barrier layer. We expect our study to provide insights on the toxicity of environmental contaminants on organisms under encapsulated conditions that could potentially be useful for toxicity screening and in evaluating ecological risk of soil, plant, and mammalian microbiome.