Biology subjects

Chen, P.-R.

Publications and source records attributed to Chen, P.-R..

4 recordsLinked to original sources

bioRxiv2025

Engineering orthogonal and synergistic modules in marine bacteria for efficient upcycling of lignin-derived carbon

Reverting waste carbon into chemicals and materials with minimal freshwater reliance is essential for a sustainable future. Here, we report the establishment of a blue biological scheme by repurposing the evolutionary fitness of marine bacterium Roseovarius nubinhibens for efficient conversion of lignin-derived carbon into valuable chemicals with seawater as a water source. We created independency of the superior catalytic capacity of R. nubinhibens to circumvent a rare polar effect, and genetically decoupled the bioproduction pathway from central metabolism, channeling all lignin-derived carbon into desired products. Next, an orthogonal growth module was generated with acetate as a second carbon source, which is a ubiquitous byproduct and inhibitor in plant biomass hydrolysis, to sustain cell growth and supply necessary resources, but not substrates, for the growth-coupled bioproduction. Besides this designed metabolic segregation, we identified a cross-module push-and-pull synergy between bioproduction and cell growth, enabling enhanced performance and robustness. By deploying this biological scheme, we successfully achieved escalated conversion of the lignin-derived monomer 4-hydroxybenzoate to value-added compounds protocatechuate and {beta}-ketoadipate with seawater, rather than freshwater, as the water source, generating a distinct blue biological scheme with potentials in simultaneous carbon and water conservation.

bioengineering↗

bioRxiv2025

Unveiling the hidden window of prime editing

Cleavage-dependent CRISPR-Cas gene editing relies on RNA-guided DNA cleavages that push cellular machinery to incorporate intended edits. Yet, a second cleavage on the already cut DNA strand can also be executed by CRISPR nucleases. This feature, however, has never been repurposed for gene editing. Here, we report the first integration of the second cleavage activity of Cas9 for precision gene editing, allowing previously impossible prime editing in the 5 direction of a nick. We elucidate the second-cleavage-driven pathway that primes non-canonical reverse transcription events upstream of the nick. We identify the competition between the non-canonical and canonical routes, which can be modulated by rationally designing RNA templates with intended edits. We demonstrate that cellular physiologies elevate editing efficiency from individual reverse-transcripts yet exert limited influence on the pathway competitions. Our findings reshape the design principle of prime editing and open an entirely new dimension for engineering CRISPR-Cas systems with the intrinsic, non-host-specific second cleavage activity.

synthetic biology↗

bioRxiv2024

Awakening prime editing for precision engineering of probiotic Escherichia coli Nissle 1917

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 and an optimized workflow. This was realized by assembling a prime editor consisting of the CRISPR-Cas system from Streptococcus pyogenes with its native codons and a codon-optimized reverse transcriptase, and by orchestrating the induction levels. 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 developed a barcoding system for strain tracking and an antibiotic-resistance-gene-free platform to enable non-natural functionalities. Our prime editing strategy awakens back-to-basics CRISPR-Cas systems devoid of complex or extraneous designs, paving the way for future innovations in engineered probiotics.

synthetic biology↗

bioRxiv2023

Hydrogen Sulfide Coordinates Glucose Metabolism Switch through Destabilizing Tetrameric Pyruvate Kinase M2

Cancer cells reprogram their glucose metabolic pathway from oxidative phosphorylation toward aerobic glycolysis. Pyruvate kinase M2 (PKM2), which converts phosphoenolpyruvate (PEP) to pyruvate, is considered the rate-limiting enzyme involved in cancer glucose metabolism. By reducing PKM2 enzyme activity, cancer cells attain a greater fraction of glycolytic metabolites for macromolecule synthesis needed for rapid proliferation. Here we demonstrate that hydrogen sulfide (H2S) destabilizes PKM2 tetramer into dimer/monomer, leading to reduced PKM2 enzyme activity and an increase in the activation of nuclear transcriptional genes mediated by dimeric PKM2. Proteomic profiling of endogenous PKM2 reveals the occurrence of sulfhydration at cysteines, notably at cysteine 326. Blocking PKM2 sulfhydration at cysteine 326 through amino acid mutation stabilizes PKM2 tetramer and crystal structure further indicating that the tetramer organization of PKM2C326S is different from the currently known T or R states, revealing PKM2C326S as a newly identified form. The presence of a PKM2C326S mutant in cancer cells effectively rewires glucose metabolism to mitochondrial respiration, resulting in the significant inhibition of tumor growth. Collectively, PKM2 sulfhydration by H2S serves as a glucose metabolic rewiring mechanism in promoting tumorigenesis, and inhibition of PKM2 sulfhydration may be applied as a new therapeutic approach targeting cancer metabolism. One-Sentence SummaryH2S rewires glucose metabolism by destabilizing PKM2 tetramerization majorly through sulfhydration at cysteine 326 HighlightsO_LIH2S enhances PKM2 dissociation from tetramer to dimer to facilitate dimeric PKM2 nuclear translocation. C_LIO_LIH2S modifies PKM2 sulfhydration, notably at cysteine 326. C_LIO_LIThe crystal structure reveals PKM2C326S as a unique tetramer conformation. C_LIO_LIBlockage of PKM2 sulfhydration at C326 rewires cancer glucose metabolism and significantly inhibits tumor growth. C_LI

cancer biology↗