Biology subjects

Lai, H.-E.

Publications and source records attributed to Lai, H.-E..

6 recordsLinked to original sources

bioRxiv2025

DiscERN: An Automated Genome Mining Tool for the Discovery of Evolutionarily Related Natural Products

Targeted genome mining to expand known families of natural products is a powerful strategy for discovering bioactive compounds, yet it remains a significant bioinformatics challenge. While tools exist for de novo biosynthetic gene cluster identification and large-scale unsupervised clustering, dedicated methods for the targeted, hypothesis-driven expansion of user-defined BGC families are lacking. Here, we present DiscERN (Discoverer of Evolutionarily Related Natural products), a userfriendly tool designed to address this gap. DiscERN leverages a multi-modal ensemble method that integrates four complementary algorithms classifying biosynthetic gene clusters based on Pfam content, sequence homology, and predicted product structure. This approach allows users to strategically balance discovery sensitivity with predictive precision to suit diverse research goals. We demonstrate DiscERNs utility by applying it to a large collection of actinomycete genomes and validating its predictive power through the successful isolation of discomycin A, a new calcium-dependent lipopeptide antibiotic, from a silent biosynthetic gene cluster. DiscERN provides a robust and accessible platform that streamlines the path from genomic data to a prioritised list of candidate biosynthetic gene clusters, effectively bridging the gap between in silico prediction and bioactive compound discovery.

bioinformatics↗

bioRxiv2024

Biosynthesis of Arcyriaflavin F from Streptomyces venezuelae ATCC 10712

Indolocarbazoles are natural products with a broad spectrum of reported bioactivities. A distinct feature of indolocarbazole biosynthesis is the modification of the indole and maleimide rings by regioselective tailoring enzymes. Here, we study a new indolocarbazole variant, which is encoded by the acfXODCP genes from Streptomyces venezuelae ATCC 10712. First, we characterise this pathway by expressing the acfXODCP genes in Streptomyces coelicolor, which led to the production of a C-5/C-5-dihydroxylated indolocarbazole. We name this new product arcyriaflavin F. Second, we demonstrate the flavin-dependent monooxygenase AcfX catalyses the C-5/C-5 dihydroxylation of the unsubstituted arcyriaflavin A into arcyriaflavin F. Interestingly, AcfX shares homology to EspX from erdasporine A biosynthesis, which instead catalyses a single C-6 indolocarbazole hydroxylation. In summary, we report a new indolocarbazole biosynthetic pathway and a regioselective C-5 indole ring tailoring enzyme AcfX. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=46 SRC="FIGDIR/small/589956v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@c0c397org.highwire.dtl.DTLVardef@16915dforg.highwire.dtl.DTLVardef@1ae538org.highwire.dtl.DTLVardef@1bef4ed_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology

Prokaryotic cell-free coupled transcription-translation (TX-TL) systems are emerging as a powerful tool to examine natural product biosynthetic pathways in a test-tube. The key advantages of this approach are the reduced experimental timescales and controlled reaction conditions. In order to realise this potential, specialised cell-free systems in organisms enriched for biosynthetic gene clusters, with strong protein production and well-characterised synthetic biology tools, is essential. The Streptomyces genus is a major source of natural products. To study enzymes and pathways from Streptomyces, we originally developed a homologous Streptomyces cell-free system to provide a native protein folding environment, a high G+C (%) tRNA pool and an active background metabolism. However, our initial yields were low (36 g/mL) and showed a high level of batch-to-batch variation. Here, we present an updated high-yield and robust Streptomyces TX-TL protocol, reaching up to yields of 266 g/mL of expressed recombinant protein. To complement this, we rapidly characterise a range of DNA parts with different reporters, express high G+C (%) biosynthetic genes and demonstrate an initial proof of concept for combined transcription, translation and biosynthesis of Streptomyces metabolic pathways in a single one-pot reaction.

synthetic biology↗

bioRxiv2019

A biosynthetic platform for antimalarial drug discovery

Advances in synthetic biology have enabled production of a variety of compounds using bacteria as a vehicle for complex compound biosynthesis. Violacein, a naturally occurring indole pigment with antibiotic properties, can be biosynthetically engineered in Escherichia coli expressing its non-native synthesis pathway. To explore whether this synthetic biosynthesis platform could be used for drug discovery, here we have screened bacterially-derived violacein against the main causative agent of human malaria, Plasmodium falciparum. We show the antiparasitic activity of bacterially-derived violacein against the P. falciparum 3D7 laboratory reference strain as well as drug-sensitive and resistant patient isolates, confirming the potential utility of this drug as an antimalarial. We then screen a biosynthetic series of violacein derivatives against P. falciparum growth. The demonstrated varied activity of each derivative against asexual parasite growth points to potential for further development of violacein as an antimalarial. Towards defining its mode of action, we show that biosynthetic violacein affects the parasite actin cytoskeleton, resulting in an accumulation of actin signal that is independent of actin polymerization. This activity points to a target that modulates actin behaviour in the cell either in terms of its regulation or its folding. More broadly, our data show that bacterial synthetic biosynthesis is a suitable platform for antimalarial drug discovery with potential applications in high-throughput and cost-effective drug screening with otherwise chemically-intractable natural products.

synthetic biology↗

bioRxiv2018

A low-cost biological agglutination assay for medical diagnostic applications

Affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing are key in addressing challenges in the diagnosis of medical conditions and for improving global health in general. Ideally, POC diagnostic tests should be highly selective for the biomarker, user-friendly, have a flexible design architecture and a low cost of production. Here we developed a novel agglutination assay based on whole E. coli cells surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design as a new diagnostic platform by the detection of a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations supported using straight-forward design rules and a mathematical model. Finally, we re-engineer E. coli cells for the detection of a medically relevant biomarker by the display of two different antibodies against the human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for both field-testing in the developing world, emergency or disaster relief sites as well as routine medical testing and personalized medicine.

synthetic biology↗

bioRxiv2017

A semi-synthetic strategy for derivatization of the violacein natural product scaffold

Natural products and their analogues are often challenging to synthesise due to their complex scaffolds and embedded functional groups. Solely relying on engineering the biosynthesis of natural products may lead to limited compound diversity. Integrating synthetic biology with synthetic chemistry allows rapid access to much more diverse portfolios of xenobiotic compounds which may accelerate the discovery of new therapeutics. As a proof-of-concept, by supplementing an Escherichia coli strain expressing the violacein biosynthesis pathway with 5-bromo-tryptophan in vitro or tryptophan 7-halogenase RebH in vivo, 6 halogenated analogues of violacein or deoxyviolacein were generated, demonstrating promiscuity of the violacein biosynthesis pathway. Furthermore, 20 new derivatives were generated from 5-brominated violacein analogues via Suzuki-Miyaura cross-coupling reaction directly using the crude extract without prior purification. Herein, we demonstrate a flexible and rapid approach to access diverse chemical space that can be applied to a wide range of natural product scaffolds.

synthetic biology↗