Biology subjects

Yuan, X.-Z.

Publications and source records attributed to Yuan, X.-Z..

5 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↗

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↗