Biology subjects

Shen, H.-W.

Publications and source records attributed to Shen, H.-W..

3 recordsLinked to original sources

bioRxiv2026

Tailoring Precise Genomic Integration Toward Isolate-to-Industry Strain Development for Scalable High-Titer Production of Polyhydroxyalkanoate

Halophilic chassis has emerged as a promising biomanufacturing platform for industrial polyhydroxyalkanoate (PHA) production. However, challenges still remain in improving the production capacity, scalability and robustness, thereby lowering cost to meet market demands. Here, a high-performing halophilic strain Halomonas LY03 was isolated with over 38% glucose- to-PHA conversion rate and broad non-grain substrate utilization capability. Multidimensional tools, including algorithm-guided high-expression neutral integration site (HENIS) screening toolkit designated SiteSeek, stop codon (TAA)-dependent enhancement of gene expression and recombinase-mediated large-fragment (> 9 kb) genomic integration, were then developed to enable precise, efficient and interference-free genomic integrative expression. Using these tools, various chromosomally engineered strains were rapidly constructed to achieve high-level production of poly-3-hydroxybutyrate (PHB, 151 g L-{superscript 1}) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB, 139 g L-{superscript 1}) under high cell-density fermentation (up to 186 g L-1 cell dry weight) in a 5-L bioreactor. Scalability was demonstrated at 2-m3 and 20-m3 industry-scale fermentations, yielding up to 134 g L-{superscript 1} PHB and 127 g L-{superscript 1} P34HB (6.1 mol% 4HB). Building on the proven robustness, a two-stage continuous fermentation (TCF) process was developed using a twin-bioreactor system at 5-L and 20-m3 scales, where stable and sustained PHA production lasted over 260 h and 160 h, respectively. Techno-economic analysis revealed a substantial cost-reduction space of 48% compared with conventional fed-batch process. This study demonstrates a successful paradigm for engineering a newly isolated strain toward robust, high-titer and cost-competitive PHA production across lab-to-industry scales.

synthetic biology↗

bioRxiv2026

Engineering xylose metabolism for diverse polyhydroxyalkanoates synthesis in Halomonas TD

Engineering the biosynthesis of fully biodegradable polyhydroxyalkanoates (PHAs) from non-food and renewable feedstocks like lignocellulose is becoming an attractive strategy for sustainable biomanufacturing. However, the efficiency and diversity of PHA synthesis from lignocellulosic hydrolysate (LH), mainly containing glucose and xylose, still remains challenge. Here, Halomonas TD, a cost-effective PHA-producing chassis, was developed to utilize glucose and xylose (or LH) for effective production of poly-3-hydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] by engineering the phosphoketolase pathway-dependent xylose metabolism in the genome, yielding 50.1 g L-1 PHB and 42.6 g L-1 P(3HB-co- 11.4 mol% 4HB) under fed-batch condition. Subsequently, the introduction of Weimberg pathway was found be able to synthesize terpolymer consisting of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate (3HV), namely [P(3HB-co-4HB-co-3HV)], from glucose and xylose only due to the isoenzyme activity of keto-acid decarboxylase encoded by kivD, which converts xylose-derived intermediate 2,5-dioxopentanoate and -ketoglutarate into butanedial (4HB synthesis) and 2-ketobutyrate (3HV synthesis), respectively. Finally, a tailored-made xylose-induced system was constructed to achieve exquisite xylose transmembrane transportation control for improved synthesis of terpolymer P(3HB-co-4.5 mol% 4HB-co- 3.0 mol% 3HV), reaching to 6.3 g L-1 under shake-flask condition, together with the co-expression of fine-tuned phosphoketolase and Weimberg pathways. This study provides a feasible and sustainable alternative for lignocellulosic resources valorization powered by the engineered Halomonas TD capable of efficient xylose utilization and diverse PHAs synthesis.

bioengineering↗

bioRxiv2021

Upregulation of the Cdc42 GTPase limits replicative lifespan in budding yeast

Cdc42, a conserved Rho GTPase, plays a central role in polarity establishment in yeast and animals. Cell polarity is critical for asymmetric cell division, and asymmetric cell division underlies replicative aging of budding yeast. Yet how Cdc42 and other polarity factors impact lifespan is largely unknown. Here, we show by live-cell imaging that the active Cdc42 level is sporadically elevated in wild type during repeated cell divisions but rarely in the long-lived bud8 deletion cells. We find a novel Bud8 localization with cytokinesis remnants, which also recruit Rga1, a Cdc42 GTPase activating protein. Genetic analyses and live-cell imaging suggest that Rga1 and Bud8 oppositely impact lifespan likely by modulating active Cdc42 levels. An rga1 mutant, which has a shorter lifespan, dies at the unbudded state with a defect in polarity establishment. Remarkably, Cdc42 accumulates in old cells, and its mild overexpression accelerates aging with frequent symmetric cell divisions, despite no harmful effects on young cells. Our findings implicate that the interplay among these positive and negative polarity factors limits the lifespan of budding yeast.

cell biology↗