Utilizing Ribose 1,5-Bisphosphate Isomerase and RuBisCO-Equipped Escherichia coli Nissle for Low-Carbon Footprint GABA Production DOI Creative Commons
Sefli Sri Wahyu Effendi, Yoshihiro Toya, Hiroshi Shimizu

et al.

ACS Sustainable Chemistry & Engineering, Journal Year: 2024, Volume and Issue: 12(30), P. 11093 - 11098

Published: July 16, 2024

Escherichia coli Nissle 1917 (EcN), the only probiotic E. coli, has been exploited as a promising chemical bioproducer due to possessing unique mutations under acidic conditions. To bolster its sustainability, novel CO2-recycling system was reconstructed by coexpressing ribose-1,5-bisphosphate isomerase (R15Pi) and ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) (i.e., RR plasmid). The function of examined through transcription level R15P-generating gene (phnN), showing higher mRNA in EcN. Afterward, RR-equipped EcN strain utilized for recycling CO2 release during γ-aminobutyric acid (GABA) synthesis, improving yield 12, 12.5, 14% assimilation glucose, acetate, glycerol medium, respectively. with low copy GadB plasmid RR+LG strain) successfully assimilated 27–37% within three mediums. artificial CO2-fixing via R15Pi RuBisCO, thus manifesting prospects low-carbon-featuring microbial cell factory new pathway.

Language: Английский

Engineering Escherichia coli Nissle 1917 as a microbial chassis for therapeutic and industrial applications DOI Creative Commons

Mingjing Yu,

Shilong Hu,

Biao Tang

et al.

Biotechnology Advances, Journal Year: 2023, Volume and Issue: 67, P. 108202 - 108202

Published: June 19, 2023

Language: Английский

Citations

30
Genetically Encoded Biosensors for Constrained Biological Functions in Probiotic Escherichia coli Nissle DOI
Shan Jiang, Haofeng Chen,

Shiyao Chen

et al.

ACS Synthetic Biology, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 8, 2025

The probiotic Escherichia coli Nissle (EcN) is an exceptional strain that has attracted significant attention not only for its clinical efficacy in the treatment and prevention of gastrointestinal disorders but also as a burgeoning microbial chassis living therapeutic applications. However, there immediate necessity to develop conditional expression systems confine activity EcN specifically tract, avoid influencing environment. Here, we constructed two genetically encoded interchangeable sensors responsive body temperature at 37 °C, small molecules such protocatechuic acid (PCA), metabolite found green tea. By employing dCpf1 targeted deactivation LacI gene, thereby coupled above sensing modules with Ptrc-lacO system achieved improved signal outputs relatively high ON/OFF ratios. Subsequently, validated biological function engineering using enhanced fluorescent protein (eGFP) animal model mice. Taken together, construction restrict functions would be applicable real-world implementation therapeutics or drug delivery.

Language: Английский

Citations

1
Minimizing endogenous cryptic plasmids to construct antibiotic-free expression systems for Escherichia coli Nissle 1917 DOI Creative Commons

Siyan Zhou,

Linlin Zhao, Wenjie Zuo

et al.

Synthetic and Systems Biotechnology, Journal Year: 2024, Volume and Issue: 9(1), P. 165 - 175

Published: Jan. 25, 2024

The probiotic bacterium Escherichia coli Nissle 1917 (EcN) holds significant promise for use in clinical and biological industries. However, the reliance on antibiotics to maintain plasmid-borne genes has overshadowed its benefits. In this study, we addressed issue by engineering endogenous cryptic plasmids pMUT1 pMUT2. non-essential elements were removed create more stable derivatives pMUT1NR△ pMUT2HBC△. Synthetic promoters integrating binding motifs sigma factors further constructed applied expression of Bacteroides thetaiotaomicron heparinase III biosynthesis ectoine. Compared traditional antibiotic-dependent systems, our newly antibiotic-free systems offer considerable advantages synthetic biology applications.

Language: Английский

Citations

6
Kinetic model assisted synthesis of β-alanine from 3-aminopropionaldehyde by a novel single step enzymatic biotransformation DOI

M. P. Karthikeyan,

Sathish Kumar Shanmugam,

Andrea R Kagoo

et al.

Biocatalysis and Biotransformation, Journal Year: 2025, Volume and Issue: unknown, P. 1 - 12

Published: May 12, 2025

Language: Английский

Citations

0
Dynamically Regulating Glucose Uptake to Reduce Overflow Metabolism with a Quorum-Sensing Circuit for the Efficient Synthesis of d-Pantothenic Acid in Bacillus subtilis DOI

Panhong Yuan,

Mengtao Xu,

Chengyao Mao

et al.

ACS Synthetic Biology, Journal Year: 2023, Volume and Issue: 12(10), P. 2983 - 2995

Published: Sept. 4, 2023

In response to a high concentration of glucose, Bacillus subtilis, microbial chassis for producing many industrial metabolites, rapidly takes up glucose using the phosphotransferase system (PTS), leading overflow metabolism, common phenomenon observed in bacteria. Although metabolism affects cell growth and reduces production effective strategies that reduce while maintaining normal remain be developed. Here, we used quorum sensing (QS)-mediated circuit tune uptake rate thereby relieve an engineered B. subtilis d-pantothenic acid (DPA). A low-efficiency non-PTS was at early stages avoid rapid glycolytic flux, efficient PTS system, which activated by QS circuit, automatically late after surpassing threshold density. This strategy successfully applied as modular metabolic engineering process DPA. By enhancing translation levels key enzymes (3-methyl-2-oxobutanoate hydroxymethytransferase, pantothenate synthetase, aspartate 1-decarboxylase proenzyme, 2-dehydropantoate 2-reductase, dihydroxy-acid dehydratase, acetolactate synthase) with 5'-untranslated regions (UTRs) mRNAs, flux promoted direction DPA production, elevating yield 5.11 g/L shake flasks. Finally, produced 21.52 fed-batch fermentations. Our work not only revealed new reducing adjusting combination promoting through 5'-UTR mRNAs but also showed its power bioproduction exhibiting promising application prospects.

Language: Английский

Citations

9
Advances in the synthesis of β-alanine DOI Creative Commons
Peng Song, Xue Zhang,

Shuhua Wang

et al.

Frontiers in Bioengineering and Biotechnology, Journal Year: 2023, Volume and Issue: 11

Published: Oct. 26, 2023

β-Alanine is the only naturally occurring β-type amino acid in nature, and it also one of very promising three-carbon platform compounds that can be applied cosmetics food additives as a precursor chemical, pharmaceutical material fields, with broad market prospects. synthesized through chemical biological methods. The synthesis method relatively well developed, but reaction conditions are extreme, requiring high temperature pressure strongly acidic alkaline conditions; moreover, there many byproducts require energy consumption. Biological methods have advantages product specificity, mild conditions, simple processes, making them more production for β-alanine. This paper provides systematic review pathways, mechanisms, key synthetic enzymes factors influencing β-alanine, view to providing reference development highly efficient green process β-alanine its industrialization, basis further innovations

Language: Английский

Citations

9
Efficient Fermentative Production of β-Alanine from Glucose through Multidimensional Engineering of Escherichia coli DOI
Yufei Zhang, Guoyan Zhang, Huifang Zhang

et al.

Journal of Agricultural and Food Chemistry, Journal Year: 2024, Volume and Issue: 72(25), P. 14274 - 14283

Published: June 13, 2024

β-Alanine, a valuable β-type amino acid, is experiencing increased demand due to its multifaceted applications in food flavoring, nutritional supplements, pharmaceuticals, and the chemical industry. Nevertheless, sustainable biosynthesis of β-alanine currently faces challenges scarcity robust strains, attributed complexities modulating multiple genes inherent physiological constraints. Here, systems metabolic engineering was implemented

Language: Английский

Citations

3
Engineering probiotic Escherichia coli Nissle 1917 to block transfer of multiple antibiotic resistance genes by exploiting a type I CRISPR-Cas system DOI
Mengdie Fang, Ruiting Zhang, Chen‐Yu Wang

et al.

Applied and Environmental Microbiology, Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 10, 2024

ABSTRACT Many multidrug-resistant (MDR) bacteria have evolved through the accumulation of antibiotic resistance genes (ARGs). Although potential risk probiotics as reservoirs ARGs has been recognized, strategies for blocking transfer while using rarely explored. The probiotic Escherichia coli Nissle 1917 (EcN) long used treating intestinal diseases. Here, we demonstrate frequent into EcN both in vitro and vivo , raising concerns about its accumulating resistance. Given that no CRISPR-Cas system was found natural EcN, integrated type I-E CRISPR-Cas3 derived from E. BW25113 EcN. engineered able to efficiently cleave multiple [i.e., mcr-1 bla NDM-1 tet (X)] encoding enzymes degrading last-resort antibiotics. Through co-incubation expressing Cas3-Cascade Cas9, showed growth former strain outcompeted latter strain, demonstrating a better clinical application prospect system. In intestine model animal (i.e., zebrafish), exhibited immunity against CRISPR-targeted ARGs. Our work equips with by exploiting exogenous system, thereby reducing spread it chassis generating living therapeutics. IMPORTANCE To reduce development resistance, considered substitute However, themselves are This study introduces new strategy limiting engineering typical (EcN), which diseases developed We also I imposes lower burden than II highlighting promising potential. not only provides restricting but enriches genetic toolbox paving way safe use

Language: Английский

Citations

3
Engineering probioticEscherichia coliNissle 1917 to block transfer of multiple antibiotic resistance genes by exploiting a type I CRISPR-Cas system DOI Creative Commons
Mengdie Fang, Ruiting Zhang, Chen‐Yu Wang

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 2, 2024

Abstract Many multidrug-resistant (MDR) bacteria evolved through accumulation of antibiotic-resistance genes (ARGs). Although the potential risk probiotics as reservoirs ARGs has been recognized, strategies for blocking transfer while using have rarely explored. The probiotic Escherichia coli Nissle 1917 (EcN) long used treating intestinal diseases. Here, we showed frequent into EcN both in vitro and vivo , raising its accumulating antibiotic resistance. Given that no CRISPR-Cas system is found natural EcN, integrated endogenous type I-E derived from E. BW25113 engineered was able to efficiently cleave multiple (i.e., mcr-1 bla NDM-1 tet (X)). By co-incubation expressing Cas3-Cascade Cas9, growth former strain outcompeted latter strain, demonstrating better clinical application prospect system. Finally, exhibited immunity against targeted intestine a model animal (i.e. zebrafish). Our work provides new strategy restricting paving way safe use this development living therapeutics.

Language: Английский

Citations

2
Development of a Type I-E CRISPR-Based Programmable Repression System for Fine-Tuning Metabolic Flux toward D-Pantothenic Acid in Bacillus subtilis DOI

Chengyao Mao,

Han Zheng, Yi‐Feng Chen

et al.

ACS Synthetic Biology, Journal Year: 2024, Volume and Issue: 13(8), P. 2480 - 2491

Published: July 31, 2024

The CRISPR-based regulation tools enable fine-tuning of gene transcription, showing potential in areas biomanufacturing and live therapeutics. However, the cell toxicity PAM specificity existing systems limit their broad application. development new less-toxic CRISPR-controlled expression remains highly desirable for expanding application scope tools. Here, we reconstituted type I CRISPR-Cas system from Escherichia coli to finely tune Bacillus subtilis. Through engineering 5′ untranslated region (UTR) mRNAs cas genes, remarkably improved efficacy CRISPRi system. was applied D-pantothenic acid (DPA)-producing B. subtilis, which generated by strengthening metabolic flux toward β-alanine (R)-pantoate via enhancing key enzymes at both transcriptional translational levels. controlling pdhA with DPA TCA cycle, elevated titer 0.88 g/L shake flasks 12.81 fed-batch fermentations without addition precursor β-alanine. strategy reported here not only enrich CRISPR toolbox subtilis facilitate production through microbial fermentation but also provide a paradigm programming important organisms produce value-added chemicals cheap raw materials.

Language: Английский

Citations

2