Biology subjects

Wong, F. T.

Publications and source records attributed to Wong, F. T..

16 recordsLinked to original sources

bioRxiv2026

MethylAmp One-step isothermal amplification with preservation of DNA methylation patterns

DNA methylation is an important epigenetic modification involved in gene regulation, genome stability, development, and disease. Analysis of low-abundance methylated DNA often requires DNA amplification. Conventional amplification methods, however, do not preserve methylation marks on newly synthesized DNA, potentially compromising downstream methylation analysis. Recently, DNA methyltransferase 1 (DNMT1) was coupled with phi29-mediated whole-genome amplification to preserve DNA methylation for downstream bisulfite sequencing. Targeted amplification of short, predefined genomic loci requires a different amplification strategy. Here, we developed MethylHDA, a one-pot isothermal workflow that integrates primer-directed helicase-dependent amplification (HDA) with DNMT1-mediated maintenance of DNA methylation. Because standard thermophilic HDA is performed at approximately 65 {degrees}C, a temperature incompatible with sustained DNMT1 activity, we established a unified reaction buffer and operating temperature of 42 {degrees}C that supported both HDA and DNMT1. Under these conditions, HDA produced an approximately five-cycle reduction in qPCR threshold cycle (Ct) relative to the unamplified control. Methylation-sensitive restriction enzyme quantitative PCR (MSRE-qPCR) demonstrated that inclusion of DNMT1 increased retention of methylation-dependent restriction protection and that the methylation signal was proportional to the methylated fraction of the input templates. These results demonstrate the feasibility of coupling targeted isothermal DNA amplification with concurrent maintenance of DNA methylation in a single reaction. This approach provides a foundation for methylation-preserving preamplification of short, locus-specific targets for downstream MSRE-qPCR and other targeted methylation assays.

molecular biology↗

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

Further characterization and engineering of a 11-amino acid motif for enhancing recombinant protein expression

BackgroundRecombinant protein production in Escherichia coli (E. coli) is a widely used system in both academic and industrial research owing to its low cost and wide availability of genetic tools. Despite its advantages, this system still struggles with soluble expression of recombinant proteins. To address this, various solubility-enhancing and yield-improving methods such as the addition of fusion tags have been developed. However, traditional tags such as small ubiquitin-related modifier (SUMO) and Glutathione S-transferase (GST) can interfere with protein folding or require removal post-translation, which adds complexity and cost to production. To circumvent these issues, smaller solubility tags (<10 kDa) are preferred. This study specifically focuses on an 11-amino acid solubility-enhancing tag (NT11) derived from the N-terminal domain of a duplicated carbonic anhydrase from Dunaliella species. ResultsA comprehensive analysis was performed to improve the characteristics of the 11-amino acid tag. By investigating the alanine-scan library of NT11, we increased its activity and identified key residues for further development. Screening with the alanine mutant library consistently led to at least a two-fold improvement in protein yield for three different proteins. We also discovered that the NT11 tag is not limited to the N-terminal position and can function at either the N- or C-terminal of the protein, providing flexibility in designing protein expression constructs. With these new insights, we have successfully doubled the recombinant protein yields of valuable growth factors, such as fibroblast growth factor 2 (FGF2) and an originally low-yielding human epidermal growth factor (hEGF), in E. coli. ConclusionThe further characterisation and development of the NT11 tag have provided valuable insights into the optimization process for such small tags and expanded our understanding of its potential applications. The ability of NT11 tag to be positioned at different locations within the protein construct without compromising its effectiveness to enhance recombinant protein yields, makes it a valuable tool across a diverse range of proteins. Collectively, these findings have the potential to simplify and enhance the efficiency of recombinant protein production.

synthetic biology↗

bioRxiv2024

Valorisation of spent cultivated meat media for recombinant FGF2 production in GRAS Lactococcus lactis

Innovative strategies for sustainable utilization of waste resources are imperative in the pursuit of a circular economy. Recently, the idea of utilizing mammalian spent media as a valuable resource is gaining traction, offering significant opportunities for innovative uses as a food-grade feedstock for microbial fermentation, especially in the production of alternative proteins for research and food purposes. In this study, we aim to repurpose spent mammalian culture media for production of valuable proteins. Growth factors (GFs) are a family of high-value proteins that naturally stimulate cell proliferation or differentiation. More importantly, these factors also present significant costs for cell culture. Here, we successfully demonstrate the use of spent mammalian culture media for the recombinant production of fibroblast growth factor 2 (FGF2-G3) in Lactococcus lactis. Bioreactor fermentation at a 1 L scale confirmed purified yields of 2.6 mg/L of recombinant FGF2-G3 using spent media. Further functional testing indicated that the recombinant FGF2-G3 can promote cell proliferation on an Anguilla japonica (Japanese eel) pre-adipocytic cell line, suggesting its potential for cultivated meat production. Based on the preliminary results of this study, our calculations indicate that fermenting 1 L spent mammalian waste could yield enough growth factors to efficiently grow approximately 52 L of cultivated meat through fermentation. This prediction emphasizes the potential of waste valorisation to sustainably produce protein, thus contributing to environmental preservation and economic viability. HighlightsO_LISpent mammalian culture media can be repurposed for microbial fermentation. C_LIO_LIGRAS L. lactis was used to express FGF2-G3 in an optimized spent media formulation. C_LIO_LIFunctional FGF2-G3 expression was demonstrated in controlled 1-L bioreactor runs. C_LI

microbiology↗

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

A Sweeter Future: Using protein language models for exploring sweeter brazzein homologs

Reducing sugar intake lowers the risk of obesity and associated metabolic disorders. Currently, this is achieved using artificial non-nutritive sweeteners, where their safety is widely debated and their contributions in various diseases is controversial. Emerging research suggests that these sweeteners may even increase the risk of cancer and cardiovascular problems, and some people experience gastrointestinal issues as a result of using them. A safer alternative to artificial sweeteners could be sweet-tasting proteins, such as brazzein, which do not appear to have any adverse health effects. In this study, protein language models were explored as a new method for protein design of brazzein. This innovative approach resulted in the identification of unexpected mutations, which opened up new possibilities for engineering thermostable and potentially sweeter versions of brazzein. To facilitate the characterization of the brazzein mutants, a simplified procedure was developed for expressing and analyzing related proteins. This process involved an efficient purification method using Lactococcus lactis (L. lactis), a generally recognized as safe (GRAS) bacterium, as well as taste receptor assays to evaluate sweetness. The study successfully demonstrated the potential of computational design in producing a more heat-resistant and potentially more palatable brazzein variant, V23.

synthetic biology↗

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↗

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↗