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Genetic Circuits for Feedback Control of Gamma-Aminobutyric Acid Biosynthesis in Probiotic Escherichia coli Nissle 1917.

ABSTRACT: Engineered microorganisms such as the probiotic strain Escherichia coli Nissle 1917 (EcN) offer a strategy to sense and modulate the concentration of metabolites or therapeutics in the gastrointestinal tract. Here, we present an approach to regulate the production of the depression-associated metabolite gamma-aminobutyric acid (GABA) in EcN using genetic circuits that implement negative feedback. We engineered EcN to produce GABA by overexpressing glutamate decarboxylase and applied an intracellular GABA biosensor to identify growth conditions that improve GABA biosynthesis. We next employed characterized genetically encoded NOT gates to construct genetic circuits with layered feedback to control the rate of GABA biosynthesis and the concentration of GABA produced. Looking ahead, this approach may be utilized to design feedback control of microbial metabolite biosynthesis to achieve designable smart microbes that act as living therapeutics.

SUBMITTER: Lebovich M

PROVIDER: S-EPMC10819706 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Genetic Circuits for Feedback Control of Gamma-Aminobutyric Acid Biosynthesis in Probiotic <i>Escherichia coli</i> Nissle 1917.

Lebovich Matthew M Lora Marcos A MA Gracia-David Jared J Andrews Lauren B LB

Metabolites 20240111 1

Engineered microorganisms such as the probiotic strain <i>Escherichia coli</i> Nissle 1917 (EcN) offer a strategy to sense and modulate the concentration of metabolites or therapeutics in the gastrointestinal tract. Here, we present an approach to regulate the production of the depression-associated metabolite gamma-aminobutyric acid (GABA) in EcN using genetic circuits that implement negative feedback. We engineered EcN to produce GABA by overexpressing glutamate decarboxylase and applied an in ...[more]

PMID: 38248847

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Project description:CRISPR-Cas systems are transforming precision medicine with engineered probiotics as next-generation diagnostics and therapeutics. To promote human health and treat disease, engineering probiotic bacteria demands maximal versatility to enable non-natural functionalities while minimizing undesired genomic interferences. Here, we present a streamlined prime editing approach tailored for probiotic Escherichia coli Nissle 1917 utilizing only essential genetic modules, including Cas9 nickase from Streptococcus pyogenes, a codon-optimized reverse transcriptase, and a prime editing guide RNA, and an optimized workflow with longer induction. As a result, we achieved all types of prime editing in every individual round of experiments with efficiencies of 25.0%, 52.0%, and 66.7% for DNA deletion, insertion, and substitution, respectively. A comprehensive evaluation of off-target effects revealed a significant reduction in unintended mutations, particularly in comparison to two different base editing methods. Leveraging the prime editing system, we inserted a unique DNA sequence to barcode the edited strain and established an antibiotic-resistance-gene-free platform to enable non-natural functionalities. Our prime editing strategy presents a CRISPR-Cas system that can be readily implemented in any laboratories with the basic CRISPR setups, paving the way for future innovations in engineered probiotics.IMPORTANCEOne ultimate goal of gene editing is to introduce designed DNA variations at specific loci in living organisms with minimal unintended interferences in the genome. Achieving this goal is especially critical for creating engineered probiotics as living diagnostics and therapeutics to promote human health and treat diseases. In this endeavor, we report a customized prime editing system for precision engineering of probiotic Escherichia coli Nissle 1917. With such a system, we developed a barcoding system for tracking engineered strains, and we built an antibiotic-resistance-gene-free platform to enable non-natural functionalities. We provide not only a powerful gene editing approach for probiotic bacteria but also new insights into the advancement of innovative CRISPR-Cas systems.

Project description:Although Escherichia coli Nissle 1917 (EcN) has been used therapeutically for over a century, the determinants of its probiotic properties remain elusive. EcN produces two siderophore-microcins (Mcc) responsible for an antagonistic activity against other Enterobacteriaceae. EcN also synthesizes the genotoxin colibactin encoded by the pks island. Colibactin is a virulence factor and a putative pro-carcinogenic compound. Therefore, we aimed to decouple the antagonistic activity of EcN from its genotoxic activity. We demonstrated that the pks-encoded ClbP, the peptidase that activates colibactin, is required for the antagonistic activity of EcN. The analysis of a series of ClbP mutants revealed that this activity is linked to the transmembrane helices of ClbP and not the periplasmic peptidase domain, indicating the transmembrane domain is involved in some aspect of Mcc biosynthesis or secretion. A single amino acid substitution in ClbP inactivates the genotoxic activity but maintains the antagonistic activity. In an in vivo salmonellosis model, this point mutant reduced the clinical signs and the fecal shedding of Salmonella similarly to the wild type strain, whereas the clbP deletion mutant could neither protect nor outcompete the pathogen. The ClbP-dependent antibacterial effect was also observed in vitro with other E. coli strains that carry both a truncated form of the Mcc gene cluster and the pks island. In such strains, siderophore-Mcc synthesis also required the glucosyltransferase IroB involved in salmochelin production. This interplay between colibactin, salmochelin, and siderophore-Mcc biosynthetic pathways suggests that these genomic islands were co-selected and played a role in the evolution of E. coli from phylogroup B2. This co-evolution observed in EcN illustrates the fine margin between pathogenicity and probiotic activity, and the need to address both the effectiveness and safety of probiotics. Decoupling the antagonistic from the genotoxic activity by specifically inactivating ClbP peptidase domain opens the way to the safe use of EcN.

Dataset Information

Genetic Circuits for Feedback Control of Gamma-Aminobutyric Acid Biosynthesis in Probiotic Escherichia coli Nissle 1917.

Publications

Genetic Circuits for Feedback Control of Gamma-Aminobutyric Acid Biosynthesis in Probiotic <i>Escherichia coli</i> Nissle 1917.

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