Biology subjects

Qiande, M.

Publications and source records attributed to Qiande, M..

3 recordsLinked to original sources

bioRxiv2025

BOTany Methods: Accessible Automation for Plant Synthetic Biology

Most members of the synthetic biology community, particularly plant scientists, lack access to liquid handling robots to scale up experiments, enhance reproducibility, and accelerate the Design, Build, Test, Learn cycle. Biofoundries enable high throughput data acquisition to train AI models and to develop new bioproducts, but they are capital-intensive to set up and not widely distributed. Entry-level, 3D-printed robots offer more affordable alternatives, but suffer from a shortage of validated protocols that can be modified without prior coding experience. To enhance access to biological automation, we developed a collection of modular BOTany Methods using Opentrons OT-2 robots to streamline the most common methods for molecular biology research and education. Our comprehensive workflow offers automation for a variety of procedures, ranging from simple but repetitive tasks (such as primer dilution and PCR setup) to more complex operations, including Plant Modular Cloning (MoClo), bacterial transformation, and plasmid extraction. Our BOTany Methods enable undergraduate students and other early career researchers to run designer experiments using table-based inputs, without editing the custom Python scripts. This pipeline enables end-to-end molecular cloning with minimal user intervention, enhancing throughput and traceability for synthetic biology applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/671538v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1b98282org.highwire.dtl.DTLVardef@2727bdorg.highwire.dtl.DTLVardef@a2157borg.highwire.dtl.DTLVardef@1305107_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Illuminating Glucomannan Synthases to Explore Cell Wall Synthesis Bottlenecks

Hemicelluloses are important dietary fibers and a key component of lignocellulosic biomass. Despite numerous observations for fluorescently tagged cellulose synthases, the subcellular journeys and biochemical activities of intracellular cellulose synthase-like enzymes such as {beta}-mannan synthases (ManS) remain largely unexplored. This study identifies C-terminal fluorescent protein tags that maintain ManS activity in the yeast to accelerate the Design, Build, Test, Learn cycles for polysaccharide biosynthesis. Using the Amorphophallus konjac ManS as a case study, we demonstrate that the enzyme co-localizes with a known yeast marker for the Golgi apparatus despite the toxic effects of plant glucomannan accumulation in Pichia pastoris. The ManS first transmembrane domain was found to be critical for the punctate localization of the enzyme, its overall expression level and its function. Additionally, we explored how fluorescently tagged ManS is influenced by genetic or chemical perturbations of native yeast cell wall components, such as reducing protein mannosylation and severely disrupting {beta}-1,3-glucans. Finally, we identified alternative feeding strategies and episomal vectors for Pichia, which were extended to Saccharomyces cerevisiae, to accelerate hemicellulose research. We propose that expanding the Plant MoClo-compatible plasmid repertoire is essential to swiftly prototype carbohydrate-active enzymes in yeast before proceeding with more time-intensive analyses in plants. Requiring only hours or days instead of weeks or months for plant transformation/regeneration, our yeast prototyping strategies can de-risk the bioengineering of carbohydrate-active enzymes.

synthetic biology↗

bioRxiv2025

Intracellular Mannanases Sustain Matrix Polysaccharide Biosynthesis

O_LIMannans with {beta}-1,4-linked backbones are common cell wall components of algae and land plants. Prior challenges to enhance {beta}-mannan content in plants point to unclear metabolic bottlenecks and the potential for hidden biosynthetic players. C_LIO_LIEndo-{beta}-MANNANASEs (MANs) are the main glycosyl hydrolases that mobilize extracellular {beta}-mannans during seed germination. However, we found that Arabidopsis man2 man5 seeds resemble {beta}-mannan biosynthetic mutants. C_LIO_LICELLULOSE SYNTHASE-LIKE A (CSLA) overexpression restored {beta}-mannan synthesis in the man double mutant and increased the distribution of crystalline polymers, but impaired the release of other mucilaginous polysaccharides. C_LIO_LIUsing yeast synthetic biology, we dissected the functional interplay of MAN enzymes with CSLAs. Intracellular MAN2 and MAN5 reduced the quantity of insoluble {beta}-mannan but elevated the content of water-soluble carbohydrates. C_LIO_LIWe propose that Arabidopsis MAN2/5, and orthologous crop enzymes with a transmembrane domain, sustain hemicellulose production in the Golgi apparatus by cleaving insoluble {beta}-mannan polymers into hydrophilic counterparts. C_LI

biochemistry↗