Biology subjects

Xia, P.-F.

Publications and source records attributed to Xia, P.-F..

9 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

Unravelling the constrained cell growth in engineered living materials

Engineered living materials (ELMs) leverage the integrative advantages of materials science and synthetic biology for advanced functionalities. Predicting and controlling cellular behavior is essential for designing and building ELMs, requiring fundamental understanding of the growth dynamics of encapsulated cells. Here, we interrogate the interference of constrained growth on the engineered functionalities and cellular physiology of cyanobacteria and unveil the dynamic interaction between cell growth and spatial confinements within photosynthetic ELMs. We observed that engineered cyanobacteria within ELMs exhibited compromised performances in growth, uptake of non-natural substrate, and synthesis of customized products, while ELMs could protect encapsulated cells from external stresses. Besides commonly accepted external influences, we identified abnormally high levels of reactive oxygen species and impaired oxygenic photosynthesis inside the cells encapsulated in ELMs. Finally, we illustrated the dynamics of cell growth within the confined spaces enveloped by the material matrices, forming clustered cell aggregates and compressed growth bubbles until the spatial limits. Our study provides a fundamental yet often overlooked connection between cellular behavior and spatial confinements, consolidating the foundation for advanced ELM innovations.

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↗

bioRxiv2024

One-for-all gene inactivation via PAM-independent base editing in bacteria

Base editing is preferable for bacterial gene inactivation without generating double strand breaks, requiring homology recombination or highly efficient DNA delivery capability. However, the potential of base editing is limited by the adjoined dependence on the editing window and protospacer adjacent motif (PAM). Herein, we report an unconstrained base editing system to enable the inactivation of any genes of interests (GOIs) in bacteria. We first employed a dCas9 derivative, dSpRY, as the effector to build a base editor with activation-induced cytidine deaminase, releasing the dependence on PAM. Then, we programmed the base editor to exclude the START codon of a GOI instead of introducing STOP codons to obtain a universal approach for gene inactivation, namely XSTART, with an overall efficiency approaching 100%. By using XSTART, we successfully manipulated the amino acid metabolisms in Escherichia coli, generating glutamine, arginine, and aspartate auxotrophic strains. The effectiveness of XSTART was also demonstrated in probiotic E. coli Nissle 1917 and photoautotrophic cyanobacterium Synechococcus elongatus, illustrating its potential in reprogramming clinically and industrially relevant chassis. To be noticed, we observed a relatively high frequency of off-target events as a trade-off for the efficacy and universality.

synthetic biology↗

bioRxiv2024

Programmable marine bacteria catalyze the valorization of lignin monomers

Efficiently converting lignin, the second most abundant biopolymer on Earth, into valuable chemicals is pivotal for a circular economy and net-zero future. However, lignin is recalcitrant to bio-upcycling, demanding innovative solutions. We report here the biological valorization of lignin-derived aromatic carbon to value-added chemicals without requesting extra organic carbon and freshwater via reprogramming the marine Roseobacter clade bacterium Roseovarius nubinhibens. We discovered the unusual catalytic advantages of this strain for the oxidation of lignin monomers and implemented a CRISPR interference (CRISPRi) system with the lacI-Ptrc inducible module, nuclease-deactivated Cas9, and programmable gRNAs. This enabled precise and efficient repression of target genes. By deploying the customized CRISPRi, we reprogrammed the carbon flux from a lignin monomer, 4-hydroxybenzoate, to achieve maximum production of protocatechuate, a pharmaceutical compound, while maintaining essential carbon for cell growth and biocatalysis. As a result, we achieved a 4.89-fold increase in protocatechuate yield with a dual-targeting CRISPRi system. Our study introduces a new-to-the-field lineage of marine bacteria and underscores the potential of blue biotechnology leveraging resources from the ocean for simultaneous carbon and water conservation.

bioengineering↗

bioRxiv2022

Base Editing for Reprogramming Cyanobacterium Synechococcus elongatus

Global climate change demands carbon-negative innovations to reduce the concentration of atmospheric carbon dioxide (CO2). Cyanobacteria can fix CO2 from the atmosphere and can be genetically reprogrammed for the production of biofuels, chemicals and food products, making an ideal microbial chassis for carbon-negative biotechnology. However, the progress seems to be slowed down due to the lagging-behind synthetic biology toolkits, especially the CRISPR-Cas-based genome-editing tools. As such, we developed a base-editing tool based on the CRISPR-Cas system and deamination for cyanobacterium Synechococcus elongatus. We achieved efficient and precise genome editing at a single-nucleotide resolution, and identified the pure population of edited cells at the first round of selection without extra segregation. By using the base-editing tool, we successfully manipulated the glycogen metabolic pathway via the introduction of premature STOP codons to inactivate the corresponding genes. We demonstrated multiplex base editing by editing two genes at once, obtaining a nearly two-fold increase in the glycogen content. We present here the first report of base editing in the phylum of cyanobacteria, and a paradigm for applying CRISPR-Cas systems in bacteria. We believe that this work will accelerate the synthetic biology of cyanobacteria and drive more innovations to alleviate global climate change.

synthetic biology↗

bioRxiv2022

Tailoring CRISPR-Cas Immunity for the Degradation of Antibiotic Resistance Genes

The evolution and dissemination of antibiotic resistance genes (ARGs) are prompting severe health and environmental issues. While environmental processes are key barriers preventing the spread of ARGs, they are often sources of ARGs at the same time, as ARGs may be required and accumulate in the biological treatment units. An upgrading of environmental biotechnology is imperative and urgent. ARGs confer antibiotic resistance based on the DNA sequences rather than the chemistry of DNA molecules. An ARG can be considered degraded if its sequence was disrupted. Therefore, we present here that CRISPR-Cas immunity, an archaeal and bacterial immune system for eliminating invading foreign DNAs, can be repurposed and tailored for the degradation of ARGs. By deploying an artificial IncP machinery, the designed system, namely VADER, can be successfully delivered via bacterial conjugation. Then, we propose a new sector for ARG degradation to be implemented as a complement to the biological units in the framework of environmental processes. In this endeavor, a prototype conjugation reactor at a 10-mL-scale was devised, and 100% of the target ARG were eliminated in the transconjugated microbes receiving VADER in the reactor. By generating a nexus of synthetic biology and environmental biotechnology, we believe that our work is not only an enterprise for tackling ARG problems but also a potential solution for managing undesired genetic materials in general in the future. ImportanceAntibiotic resistance has been causing severe health problems and leading to millions of deaths in recent years. Environmental processes, especially the wastewater treatment sector, are important to barrier the spread of antibiotic resistance from the pharmaceutical industry, hospitals, or civil sewage. However, they have been identified as the source of antibiotic resistance at the same time, as antibiotic resistance with its main cause antibiotic resistance genes (ARGs) may be required and accumulate in the biological treatment units, leading to the dissemination of ARGs. Here, we transplanted the CRISPR-Cas system, an immune system via programmable DNA cleavage, to environmental biotechnology for tackling the antibiotic resistance dilemma thereof, and we propose a new sector in environmental processes specialized in ARG removal with a reactor inhabiting the CRISPR-Cas system per se. Our study provides a new angle to resolve public health issues via the implementation of synthetic biology at the process level.

synthetic biology↗

bioRxiv2020

Reprogramming acetogenic bacteria with CRISPR-targeted base editing via deamination

Acetogenic bacteria are rising in popularity as chassis microbes in biotechnology due to their capability of converting inorganic one-carbon (C1) gases to organic chemicals. To fully uncover the potential of acetogenic bacteria, synthetic-biology tools are imperative to either engineer designed functions or to interrogate the physiology. Here, we report a genome-editing tool at a one-nucleotide resolution, namely base editing, for acetogenic bacteria based on CRISPR-targeted deamination. This tool combines nuclease deactivated Cas9 with activation-induced cytidine deaminase to enable cytosine-to-thymine substitution without DNA cleavage, homology-directed repair, and donor DNA, which are generally the bottlenecks for applying conventional CRISPR-Cas systems in bacteria. We designed and validated a modularized base-editing tool in the model acetogenic bacterium Clostridium ljungdahlii. The editing principles were investigated, and an in-silico analysis revealed the capability of base editing across the genome. Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux towards improved acetate production. This resulted in engineered C. ljungdahlii strains with the desired phenotypes and stable genotypes. Our base-editing tool promotes the application and research in acetogenic bacteria and provides a blueprint to upgrade CRISPR-Cas-based genome editing in bacteria in general. SignificanceAcetogenic bacteria metabolize one-carbon (C1) gases, such as industrial waste gases, to produce fuels and commodity chemicals. However, the lack of efficient gene-manipulation approaches hampers faster progress in the application of acetogenic bacteria in biotechnology. We developed a CRISPR-targeted base-editing tool at a one-nucleotide resolution for acetogenic bacteria. Our tool illustrates great potential in engineering other A-T-rich bacteria and links designed single-nucleotide variations with biotechnology. It provides unique advantages for engineering industrially relevant bacteria without creating genetically modified organisms (GMOs) under the legislation of many countries. This base-editing tool provides an example for adapting CRISPR-Cas systems in bacteria, especially those that are highly sensitive to heterologously expressed Cas proteins and have limited ability of receiving foreign DNA.

synthetic biology↗