Biology subjects

Tan, G.-Y.

Publications and source records attributed to Tan, G.-Y..

3 recordsLinked to original sources

bioRxiv2025

Enhanced Hydrogen Production Through Two-Stage Fermentation Coupling Clostridium pasteurianum and Rhodobacter sphaeroides

Biological H2 production via dark fermentation is constrained by low yields and inhibitory metabolite accumulation. Coupling dark and photo fermentation effectively overcomes these issues by enhancing substrate utilization and reducing wastewater pollution, yet scalable systems for industrial application remain rare. This study presents a 10-L two-stage fermentation system (5L dark/5L photo bioreactors) using Clostridium pasteurianum DSM 525 and Rhodobacter sphaeroides ZX-5. Dark fermentation generated 3372 mL H2 from glucose, yielding effluent with 1.29 g/L acetic and 3.11 g/L butyric acids. After centrifugation, pH adjustment, and clinoptilolite deamination ([≥]60% efficiency), ammonium was reduced to <100 mg/L. The pretreated effluent, supplemented with concentrated RCVBN medium, served as photo-fermentation substrate. A butyric acid feeding strategy extended the process by 120 h, boosting H2 yield by 16.4%. The coupled system produced 6480 mL H2 (80% increase), degrading 50% acetic acid and 42% butyric acid. This work demonstrates a scalable bioreactor configuration integrating efficient biohydrogen production with value-added wastewater treatment for industrial bioenergy applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/694164v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1f324a4org.highwire.dtl.DTLVardef@1eab711org.highwire.dtl.DTLVardef@98e50dorg.highwire.dtl.DTLVardef@1e5f02e_HPS_FORMAT_FIGEXP M_FIG Graphic abstract C_FIG

bioengineering↗

bioRxiv2025

Enhancing and stabilizing a high-yield industrial Rhodobacter sphaeroides strain for coenzyme Q10 production

Strengthening high-yield phenotypes while maintaining physiological and genetic stability presents a significant challenge in the improvement of high-yield industrial strains (HIS). Coenzyme Q10 (CoQ10), a crucial quinone electron carrier in the electron transport chain, is widely used in the prevention and treatment of cardiovascular diseases. In this study, the established HIS Rhodobacter sphaeroides HY01, employed for CoQ10 production, was engineered to enhance productivity while ensuring strain stability. Comparative omics identified the PrrAB two-component system as an oxygen-responsive regulator that links CoQ10 biosynthesis to photosynthetic pathways. Mutagenesis of PrrA, guided by AlphaFold3 modeling and fluorescence screening, introduced mutations that led to a 37.5% increase in CoQ10 production. To address phenotypic reversion due to metabolic burden, genome-scale CRISPR interference (CRISPRi) screening identified key genes involved in DNA repair and stress adaptation. Deletions of these genes generated a stable strain that achieved 3.6 g/L CoQ10 in a 50- L pilot-scale fed-batch fermentation, surpassing previous reports. This study reveals PrrAB-mediated flux partitioning for redox homeostasis and provides a framework for stabilizing burdened phenotypes in photosynthetic microbes, advancing the sustainable production of redox-active metabolites. Bullet pointsO_LIIdentified the PrrAB two-component system as a critical global regulator of CoQ10 biosynthesis in Rhodobacter sphaeroides. C_LIO_LIPrrA was evolved through fluorescence-based screening and rational protein engineering, significantly enhancing CoQ10 biosynthesis in industrial high-yield strain. C_LIO_LIGenome-scale CRISPRi screening identified genes affecting R. sphaeroides HY01 stability enabling targeted modifications to stabilize high-yield CoQ10 phenotype. C_LIO_LIAchieved record 3.6 g/L CoQ10 yield in 50-L pilot-scale bioreactors enhancing microbial productivity stabilizing high-yield phenotypes advancing strain engineering. C_LI

microbiology↗

bioRxiv2020

Simple cloning of large natural product biosynthetic gene cluster by CRISPR/Cas12a-mediated fast direct capturing strategy

Directly cloning of biosynthetic gene clusters (BGCs) from microbial genomes has been revolutionizing the natural product-based drug discovery. However, it is still very challenging to efficiently clone, for example, large (> 80kb) and GC-rich (> 70%), streptomycete originating BGCs. In this study, we developed a simple, fast yet efficient and low-cost in vitro platform for direct cloning large BGCs from streptomycete genomic DNA, named as CAT-FISHING (CRISPR/Cas12a- and Agarose plug-based sysTem for Fast bIoSyntHetIc geNe cluster cloninG), by combining the advantages of CRISPR/Cas12a cleavage and bacterial artificial chromosome (BAC) library construction. CAT-FISHING was demonstrated by directly cloning large DNA fragments ranging from 47 to 139 kb with GC content of > 70% from the S. albus J1074 genome in a relatively efficient manner. Moreover, surugamides, encoded by a captured 87-kb BGC with GC content of 76%, was heterologously expressed in a Streptomyces chassis. These results indicate that CAT-FISHING is a powerful platform for BGCs batch cloning, which would be greatly beneficial to the natural products-based drug discovery. We believe that this system will lead a renaissance of interest in microorganisms as a source for drug development.

synthetic biology↗