Biology subjects

Lim, Y. H.

Publications and source records attributed to Lim, Y. H..

14 recordsLinked to original sources

bioRxiv2025

Enhanced Medium-Chain Fatty Acid Production in Megasphaera elsdenii via Adaptive Laboratory Evolution and Genomic Insights

The shift toward a sustainable bioeconomy has increased interest in microbial platforms capable of producing valuable fatty acids. Megasphaera elsdenii is emerging as a promising candidate for fatty acid bioproduction, although its performance varies depending on the strain. This study compares two M. elsdenii strains, JCM 1772 (ATCC 25940) and JCM 35779, revealing that JCM 35779 produces up to 5.8 times more hexanoic acid than JCM 1772. Comparative genomic analysis identified key differences between the strains, including the loss of enzymes involved in the pyruvate-to-butyric acid pathway in JCM 35779, indicating a rerouting of carbon flux that favors chain elongation toward hexanoic acid instead of butyric acid. Overall, this work links genomic variation to metabolic phenotype, establishes JCM 35779 as a superior medium-chain fatty acid producer, and supports the development of M. elsdenii as an effective platform for sustainable production of medium-chain fatty acids. HighlightsO_LIPig-feces M. elsdenii shows >5 times higher HA production than its rumen strain. C_LIO_LIGenomic analysis reveals strain-specific deletions in pathways for MCFA synthesis. C_LIO_LIThis includes etfB2, cat1 and abfD. C_LI

microbiology↗

bioRxiv2025

Production of Glycolic acid through Whole-Cell Bioconversion from PET Monomer Ethylene Glycol Using Engineered Corynebacterium glutamicum

In the last decade, the global warming and plastic pollution issue have driven research on developing a more sustainable platform for chemicals production from alternative feedstocks. Ethylene glycol (EG), a monomer of polyethylene terephthalate (PET) plastic, has a potential to become a renewable substrate for microbial production of value-added chemicals. This study presents a biotransformation platform using Corynebacterium glutamicum to produce glycolic acid (GA) from EG. C. glutamicum was engineered to express a heterologous EG oxidation pathway. Subsequent promoter engineering yielded strain FA4, producing 10.6 g/L GA from EG in 48 h. Implementation of a two-stage biotransformation strategy using resting cells further enhanced the GA production, reaching a cumulative GA titer of 98.8 g/L after a 72-h production. Finally, applying this platform to a simulated EG mixture from PET-degradation achieved a cumulative GA titer of 67.3 g/L over 72 h, highlighting the potential for valorizing plastic waste through this biotransformation platform. These findings establish C. glutamicum as an efficient biotransformation chassis for sustainable GA production from EG and offer a promising route for PET waste valorization into value-added chemicals.

synthetic biology↗

bioRxiv2025

A Singapore-centric Fungal Dataset of 518 Cultivated Strains with Visual Phenotypes and Taxonomic Identity

The fungal kingdom represents a greatly untapped resource to produce a wide range of bioactive secondary metabolites, including antibiotics, anticancer agents, industrially significant dyes and enzymes. To-date, it is estimated only less than 5% of all fungi have been characterised, a deficit that is especially pronounced in tropical regions like Singapore, where fungal diversity remains underexplored compared to northern hemisphere counterparts. This underlines the urgency and importance of our research which motivated the creation of our curated dataset, aiming to address this gap and contribute to understanding the broader ecosystem. We developed a generalisable cultivation workflow that enables systematic strain preparation, supports high-resolution imaging, and yields sufficient fungal biomass amenable for genomic analyses. This resulted in a diverse collection of 518 phylogenetically and ecologically varied fungal strains from both terrestrial and marine environments in biodiverse Singapore. The curated dataset from this project captures both taxonomic identity and colony-level morphological traits serving as a foundation for visual phenotype to taxonomy mapping through the integration of computer vision.

microbiology↗

bioRxiv2025

Unified Genomic and Chemical Representations Enable Bidirectional Bio-synthetic Gene Cluster and Natural Product Retrieval

Natural product discovery is increasingly driven by the ability to analyze microbial genomes for biosynthetic gene clusters (BGCs) that encode secondary metabolites. While existing approaches have successfully linked BGCs to broad classes of chemical products, they typically operate in a single modality (genomic or chemical) limiting the scope of bidirectional prediction. In this work, we propose a multimodal framework that integrates genomic and chemical information by projecting embeddings derived from pretrained language models into a common representation space. We embed genomic sequences using a BGC foundation model and represent molecules through a chemical language model, then use a metric learning model to co-embed BGCs and their associated chemical structures. This co-embedding space allows us to quantify the similarity between BGCs and compounds using similarity measures, enabling both forward and inverse retrieval tasks. Beyond retrieval, we show that the shared space can guide strain selection for targeted compounds. By identifying BGCs closest to a query compound in the embedding space, we prioritize microbial strains that encode similar clusters, thereby streamlining genome mining and retrobiosynthetic design efforts. This approach represents a generalizable, scalable strategy to bridge biological and chemical modalities in natural product discovery.

synthetic biology↗

bioRxiv2025

An Experimental and Multiphysics Simulations Study of Clostridium carboxidivorans sp. 624 for Acid and Alcohol Production in CO2/H2

Biotechnological advances in CO2 utilization through gas fermentation offer a sustainable alternative to energy-intensive chemical processes. This study investigates and optimizes fermentation dynamics of Clostridium carboxidivorans sp. 624 for enhanced production of C2-C6 acids via combined experimental and computational methods. A systematic experimental evaluation of temperature and medium conditions, along with time-course analysis elucidates metabolic pathway regulation. Experimental optimisation, complemented by modelling, identified gas-liquid volumetric ratios VL/VG = 4 as optimal for maximizing longer-chain acids production, attributed to enhanced gas solubility and substrate bioavailability. Additionally, a hybrid model combining dynamic-kinetic model and computational fluid dynamics (CFD) successfully predict product formation. By capturing spatial inhomogeneities in gas-liquid interactions, the model also provides critical insights for optimizing fermentation performance and establishes a framework for improving process design parameters for CO2/H2 fermentation, paving the way for scalable and efficient CO2-based bioprocesses.

microbiology↗

bioRxiv2025

A linked Genomics Sequencing and Mass Spectrometry multi-modal dataset and models for Streamlined Natural Products Discovery in Microbial Strain Libraries

An integrated multi-modal characterization of a microbial strain library streamlines the effort for natural product discovery. By integrating language- and transformer-based models to cross-validate mass spectrometry (MS)-genome datasets, microbial producers of diverse natural products are rapidly identified with high (75-100%) precision. Our findings demonstrate the transformative potential of linked MS-genome datasets at the strain-level to significantly accelerate discovery and enhance our understanding of microbes beyond currently known and curated knowledge.

synthetic biology↗

bioRxiv2024

Hyperporous encapsulation of microbes for whole cell biocatalysis and biomanufacturing

Compared to traditional synthetic chemical processes, biocatalysts offer a more sustainable and eco-friendly approach to producing complex molecules. In particular, whole-cell biocatalysts boast numerous advantages, including scalable, self-containing co-factor recycling systems, the use of cost-effective raw materials, and reduced purification costs. However, challenges arise when working with microbial consortia for biotransformation cascades. Our encapsulation strategy addresses these challenges by controlling microbial cell populations through physical constraints, offering a promising approach in biomanufacturing. In this work, we describe the immobilization of cells in a hyper-porous hydrogel block, which provides ample nutrient access while simplifying media changes. We encapsulated E. coli cells in a hydrogel matrix with suitable mechanical properties, effectively limiting their proliferation while sustaining recombinant GFP production. Furthermore, we successfully maintained different microbial strains spatially in a single porous hydrogel block for at least 10 days, demonstrating the potential of this method for achieving stable co-culture. Finally, we demonstrated the application of immobilized E. coli for co-culture fermentation. The immobilization of E. coli heterologously expressing RadH halogenase significantly improved the efficiency of genistein halogenation in a co-culture with genistein-producing Streptomyces compared to its non-immobilized counterpart.

bioengineering↗

bioRxiv2023

Expression and engineering of unexplored PET degrading enzymes from Microbispora, Nonomuraea, Micromonospora genus

Low recycling rates have resulted in the alarming rate of accumulation of a widely used plastic material, polyethylene terephthalate (PET). With the build-up of plastics in our environment, there is an urgent need to source for more sustainable solutions to process them. Biological methods such as enzyme-catalyzed PET recycling or bioprocessing are seen as a potential solution to this problem. Actinobacteria, known for producing enzymes involved in the degradation of complex organic molecules, are of particular interest due to their potential to produce PET degrading enzymes. The highly thermostable enzyme, leaf-branch compost cutinase (LCC) found in Actinobacteria is one such example. This work expands on the discovery and characterization of new PET degrading enzymes from Microbispora, Nonomuraea, and Micromonospora genus. Within this genus, we analyzed enzymes from the polyesterase-lipase-cutinase family, which have [~]60% similarity to LCC, where one of the enzymes was found to be capable of breaking down PET and BHET at 45-50 {degrees}C. Moreover, we were able to enhance the enzymes depolymerization rate through further engineering, resulting in a two-fold increase in activity. IMPORTANCEThe proliferation of PET plastic waste poses a significant threat to human and environmental health, making it an issue of increasing concern. In response to this challenge, scientists are investigating eco-friendly approaches, such as bioprocessing and microbial factories, to sustainably manage the growing quantity of plastic waste in our ecosystem. Despite the existence of enzymes capable of degrading PET, their scarcity in nature limits their applicability. The objective of this study is to enhance our understanding of this group of enzymes by identifying and characterizing novel ones that can facilitate the breakdown of PET waste. This data will expand the enzymatic repertoire and provide valuable insights into the prerequisites for successful PET degradation.

microbiology↗

bioRxiv2023

Design-Rules for Stapled Alpha-Helical Peptides with On-Target In Vivo Activity: Application toMdm2/X dual antagonists

Stapled -helical peptides can bind to and modulate historically intractable targets while addressing the traditional liabilities associated with peptide therapeutics. However, their pipeline advancement has been impeded by the challenges of identifying peptides with sufficient cellular uptake to engage the target protein while lacking off-target toxicities. Here, we advance the field to arrive at a workflow for identifying advanced stapled peptide lead molecules with on-target in vivo activity with no off-target cell proliferation effects. Specifically, we generated a >350-member library based on ATSP-7041, a stapled peptide Mdm2(X) antagonist with validated on-target cellular effects but with significant off-target activity. Key insights from library analysis include 1) a clear correlation between lipophilicity and permeability, 2) removal of positive charge to avoid off-target toxicities, 3) judicious placement of anionic residues to enhance peptide solubility/behavior, 4) optimization of C-terminal length and helicity to enhance cell activity, 5) optimization of staple type/number to avoid polypharmacology. Incorporation of one or more of these attributes led to molecules with improved in vitro and in vivo activities (up to a >292x improved cell proliferation EC50). A subset of peptides were devoid of off-target cell proliferation effects in cell lines lacking wild-type p53 protein (up to a >3800x on-target index). This latter improvement contrasted with clinical Mdm2 antagonistic molecules. Application of these design rules to a distinct Mdm2(X) peptide series resulted in rapid improvement in cellular activity (>150x) and removal of off-target toxicities. Overall, the detailed workflow outlined here should help researchers identify stapled -helical peptides for therapeutic impact.

biochemistry↗

bioRxiv2022

Cost-effective hybrid long-short read assembly delineates alternative GC-rich Streptomyces chassis for natural product discovery

With the advent of rapid automated in silico identification of biosynthetic gene clusters (BGCs), genomics presents vast opportunities to accelerate natural product (NP) discovery. However, prolific NP producers, Streptomyces, are exceptionally GC-rich (>80%) and highly repetitive within BGCs. These pose challenges in sequencing and high-quality genome assembly which are currently circumvented via intensive sequencing. Here, we outline a more cost-effective workflow using multiplex Illumina and Oxford Nanopore sequencing with hybrid long-short read assembly algorithms to generate high quality genomes. Our protocol involves subjecting long read-derived assemblies to up to 4 rounds of polishing with short reads to yield accurate BGC predictions. We successfully sequenced and assembled 8 GC-rich Streptomyces genomes whose lengths range from 7.1 to 12.1 Mb at an average N50 of 5.9 Mb. Taxonomic analysis revealed previous misrepresentation among these strains and allowed us to propose a potentially new species, Streptomyces sydneybrenneri. Further comprehensive characterization of their biosynthetic, pan-genomic and antibiotic resistance features especially for molecules derived from type I polyketide synthase (PKS) BGCs reflected their potential as NP chassis. Thus, the genome assemblies and insights presented here are envisioned to serve as gateway for the scientific community to expand their avenues in NP discovery. Graphic abstractSchematic of hybrid long- and short read assembly workflow for genome sequencing of GC-rich Streptomyces. Boxes shaded blue and grey correspond to experimental and in silico workflows, respectively. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/519232v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1395af0org.highwire.dtl.DTLVardef@81798forg.highwire.dtl.DTLVardef@539c92org.highwire.dtl.DTLVardef@14c366f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA cost-effective genome sequencing approach for GC-rich Streptomyces is presented C_LIO_LIHybrid assembly improves BGC annotation and identification C_LIO_LIA new species, Streptomyces sydneybrenneri, identified by taxonomic analysis C_LIO_LIGenomes of 8 Streptomyces species are reported and analysed in this study C_LI

genomics↗

bioRxiv2021

Application of Cas12j for Streptomyces editing and cluster activation

In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are also oftentimes strain dependent, subsequently a variety of Cas proteins would allow for flexibility and enable genetic manipulation within a wider range of Streptomyces strains. In this work, we have further expanded the Cas toolbox by presenting the first example of Cas12j mediated editing in Streptomyces sp. A34053. In our study, we have also observed significantly improved editing efficiencies with Acidaminococcus sp. Cas12j compared to Cas12a, Francisella tularensis subsp. novicida U112s type V-A Cas (FnCpf1).

microbiology↗

bioRxiv2021

Topographic connectivity and cellular profiling reveal detailed input pathways and functionally distinct cell types in the subthalamic nucleus

The subthalamic nucleus (STN) controls psychomotor activity and is an efficient therapeutic deep brain stimulation target in Parkinsons disease patients. Despite evidence indicating position-dependent therapeutic effects and distinct functions within the STN, input circuit and cellular profile in the STN remain largely unclear. Using advanced neuroanatomical techniques, we constructed a comprehensive connectivity map of the indirect and hyperdirect pathways in both the mouse and human STN. Our detailed circuit- and cellular-level connectivity revealed a topographically graded organization with three convergent types of indirect and hyperdirect-pathways. Furthermore, we identified two functional types of glutamatergic STN neurons (parvalbumin, PV +/- neurons) segregated with a topographical distribution. Glutamatergic PV+ STN neurons contribute to burst firing. We confirmed synaptic connectivity from indirect and hyperdirect pathways to both PV+ and PV-. These data suggest a complex interplay of information integration within the basal ganglia underlying coordinated movement control and therapeutic effects.

neuroscience↗

bioRxiv2019

Biosynthetic engineering of the antifungal, anti-MRSA auroramycin

Using an established CRISPR-Cas mediated genome editing technique for streptomycetes, we explored the combinatorial biosynthesis potential of the auroramycin biosynthetic gene cluster in Streptomyces roseoporous. Auroramycin is a potent anti-MRSA polyene macrolactam. In addition, it also displays antifungal activities, which is unique among structurally similar polyene macrolactams, such as incednine and silvalactam. In this work, we employed different engineering strategies to target glycosylation and acylation biosynthetic machineries within its recently elucidated biosynthetic pathway. Six auroramycin analogs with variations in C-, N-methylation, hydroxylation and extender units incorporation were produced and characterized. By comparing the bioactivity profiles of these analogs, we determined that unique disaccharide motif of auroramycin is essential for its antimicrobial bioactivity. We further demonstrated that C-methylation of the 3, 5-epi-lemonose unit, which is unique among structurally similar polyene macrolactams, is key to its antifungal activity.

bioengineering↗

bioRxiv2019

Characterization of Cas proteins for CRISPR-Cas editing in streptomycetes

Application of the well-characterized Streptococcus pyogenes CRISPR-Cas9 system in actinomycetes has enabled high efficiency multiplex genome editing and CRISPRi-mediated transcriptional regulation in these prolific bioactive metabolite producers. Nonetheless, SpCas9 has its limitations and can be ineffective depending on the strains and target sites. Here, we built and tested alternative CRISPR-Cas constructs based on the standalone pCRISPomyces-2 editing plasmid. We showed that Streptococcus thermophilus CRISPR1 (sth1Cas9), Staphylococcus aureus Cas9 (saCas9), and Francisella tularensis subsp. Novicida U112 Cpf1 (fnCpf1) are functional in multiple streptomycetes enabling efficient homology directed repair (HDR)-mediated knock-in and deletion. In strains where spCas9 was non-functional, these alternative Cas systems enabled precise genomic modifications within biosynthetic gene clusters for the discovery, production and diversification of natural products. These additional Cas proteins provide us with the versatility to overcome the limitations of individual CRISPR-Cas systems for genome editing and transcriptional regulation of these industrially important bacteria.

synthetic biology↗