Biology subjects

Huang, Y.

Publications and source records attributed to Huang, Y..

At least 379 records · Page 21Linked to original sources

bioRxiv2024

Alteration of skin fibroblast steady state contributes to healing outcomes

Fibroblasts display complex functions associated with distinct gene expression profiles that influence matrix production and cell communications and the autonomy of tissue development and repair. Thrombospondin-2 (TSP-2), produced by fibroblasts, is a potent angiogenesis inhibitor and negatively associated with tissue repair. Single-cell (sc) sequencing analysis on WT and TSP2KO skin fibroblasts demonstrate distinct cell heterogeneity. Specifically, we found an enrichment of Sox10+ multipotent progenitor cells, identified as Schwann precursor cells, in TSP2KO fibroblasts, while fibrosis-related subpopulations decreased. Immunostaining of tissue and cells validated the increase of this Sox10+ population in KO fibroblasts. Furthermore, in silico analysis suggested enhanced pro-survival signaling, including WNT, TGF-{beta}, and PDGF-{beta}, alongside a reduced BMP4 response. Additionally, the creation of two TSP2KO NIH3T3 cell lines using the CRISPR/Cas9 technique allowed functional and signaling validation in a less complex system. Moreover, KO 3T3 cells exhibited enhanced migration and proliferation, with elevated levels of pro-regenerative molecules including TGF-{beta}3 and Wnt4, and enrichment of nuclear {beta}-catenin. These functional and molecular alterations likely contribute to improved healing and increased neurogenesis in TSP2-deficient wounds. Overall, our findings describe the heterogeneity of dermal fibroblasts and identify pro-regenerative features of TSP2KO fibroblasts.

pathology↗

bioRxiv2024

Rational molecular design for improved ZHD101 thermal stability based on the introduction of disulfide bonds at the dimer interface and B-factor analysis

AbstractZearalenone hydrolase 101 (ZHD101), derived from Clonostachys rosea, is known to effectively degrade the main contaminant (zearalenone) in animal feed, but the thermal instability of ZHD101 limits its industrial application. In this study, we successfully enhanced the thermal stability of ZHD101 through two iterative rounds of rational molecular design. First, after the prediction of disulfide bond sites, ZHD101T229C was obtained, and a new disulfide bond was formed between two single ZHD101 subunits to construct a dimer ZHD101. Then, based on ZHD101T229C, two high-vibration hotspot amino acids N137 and D170 were selected by analyzing atomic position fluctuations and dynamic information A small and precise mutant library containing three mutants (ZHD101T229C/N137L, ZHD101T229C/D170L, and ZHD101T229C/D170C) was obtained by saturation mutagenesis and calculation of binding energy in silico. Compared with the wild type, the thermal half-inactivation temperature (T50) of ZHD101T229C/D170C increased by 7{degrees}C, its half-life (t1/2) increased by 200% at 50{degrees}C, and its melting temperature (Tm) increased by 18.1{degrees}C. Molecular docking suggested that new covalent bond formation and shorter bond distance may contribute to the improved thermal stability of ZHD101. The rational design strategy proposed in this work can provide a reference for the thermal stability modification and optimization of other proteins. ImportanceZearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin that poses a significant threat to animal feed safety. ZEN hydrolase 101 (ZHD101) catalyzes the conversion of ZEN into a nontoxic product, offering a promising detoxification strategy. However, the thermal instability of ZHD101 severely limits its industrial application. In this study, the thermal stability of ZHD101 was significantly improved through two rounds of rational design, resulting in the ZHD101T229C/D170C mutant. ZHD101T229C/D170C exhibited the improved half-inactivation temperature (T50), half-life (t1/2) and the highest melting temperature (Tm) reported thus far. Overall, the iterative combinatiorial mutation strategy involved the introduction of a disulfide bond between two single subunits and the application of B-factor analysis to identify hotspot amino acids residues. This approach effectively enhanced the enzymes structural stability, paving the way for its industrial application. ZHD101T229C/D170C and the rational design strategy presented in this work provide a robust framework for the thermal stability optimization of similar biocatalysts, advancing the practical use of ZHD101 in the feed industry.

molecular biology↗

bioRxiv2024

Optimizing a Human Monoclonal Antibody for Better Neutralization of SARS-CoV-2

SARS-CoV-2 has largely evolved to resist antibody pressure, with each successive viral variant becoming more and more resistant to serum antibodies in the population. This evolution renders all previously authorized anti-spike therapeutic monoclonal antibodies inactive, and it threatens the remaining pipelines against COVID-19. We report herein the isolation of a human monoclonal antibody with a broad but incomplete SARS-CoV-2 neutralization profile, but structural analyses and mutational scanning lead to the engineering of variants that result in greater antibody flexibility while binding to the viral spike. Three such optimized monoclonal antibodies neutralize all SARS-CoV-2 strains tested with much improved potency and breadth, including against subvariants XEC and LP.8.1. The findings of this study not only present antibody candidates for clinical development against COVID-19, but also introduce a unique engineering approach to improve antibody activity via increasing conformational flexibility.

microbiology↗

bioRxiv2024

Insights into Intestinal Barrier Disruption During Long-Term Gut Chlamydia Colonization in Mice: A Single-Cell Transcriptomic Approach

Chlamydia trachomatis, an intracellular pathogen, stands as the most prevalent sexually transmitted bacterial infection among women globally. Traditionally recognized as a genital pathogen, recent research indicates that the gastrointestinal tract may also act as a reservoir for its long-term colonization. However, the mechanisms underlying Chlamydias ability to persist in the gut remain poorly understood. This gap in knowledge limits our ability to develop effective treatments for persistent Chlamydia infections. In this study we utilized single-cell RNA sequencing to analyze the gene expression profiles and cellular heterogeneity of mouse colonic tissues during Chlamydia long-term infection. This approach provided detailed insights into the transcriptional changes and cellular interactions involved in the persistence of Chlamydia in the gut. Our results revealed significant alterations in the gene expression profiles of various intestinal cell populations, with distinct molecular pathways contributing to Chlamydia persistence. Notably, we observed a reduction in the expression of markers associated with epithelial tight junctions, indicating a potential breakdown of the intestinal epithelial barrier. This impairment may facilitate the penetration of Chlamydia into deeper tissues and contribute to the initiation of infection. We also found dysregulation of the transcriptional networks in goblet cells and an imbalance in communication between immune and epithelial cells. These disruptions were linked to the pathogens ability to establish persistent colonization and infection. IMPORTANCEFew studies have explored Chlamydia persistence in the gastrointestinal tract. In this study, we use single-cell RNA sequencing to identify the molecular and cellular mechanisms driving the pathogens long-term colonization. Our findings provide crucial insights into how Chlamydia overcomes the hosts immune defenses and epithelial barriers to establish chronic infection in the gut. Notably, we identify disruption of epithelial tight junctions and an imbalance in immune-cell interactions, offering new avenues for therapeutic interventions aimed at restoring mucosal integrity and preventing persistent infection.

microbiology↗

bioRxiv2024

The high quality Chinese white truffle genome and novel fossil-calibrated estimate of Pezizomycetes divergence reveal the tempo and mode of true truffles genome evolution

The genus Tuber (family: Tuberaceae) includes the most economically valuable ectomycorrhizal (ECM), truffle-forming fungi. Previous genomic analyses revealed that massive transposable element (TE) proliferation represents a convergent genomic feature of mycorrhizal fungi, including Tuberaceae. Repetitive sequences are one of the major drivers of genome evolution shaping its architecture and regulatory networks. In this context, Tuberaceae represent an important model system to study their genomic impact; however, the family lacks high-quality assemblies. Here, we tested the interplay between TEs and Tuberaceae genome evolution by producing a highly contiguous assembly for the endangered Chinese truffle Tuber panzhihuanense, along with a novel timeline for Tuberaceae diversification and comprehensive comparative genomic analyses. We found that concurrently with a Paleogene diversification of the family, pre-existing Chromoviridae-related Gypsy clades independently expand in different truffle lineages leading to increased genome size and high gene family turnover rates, but without resulting in highly scrambled genomes. Additionally, we found an enrichment of ECM-induced gene families among ancestral duplication events. Finally, we explored the repetitive structure of nuclear ribosomal DNA (rDNA) loci for the first time in the clade. We found that most of the 45S rDNA paralogues are undergoing concerted evolution, though an isolated divergent locus raises concerns about potential issues for metabarcoding and biodiversity assessments. Our study provides a fundamental genomic resource for future research on truffle genomics and showcases a clear example on how establishment and self-perpetuating expansion of heterochromatin can drive massive genome size variation due to activity of selfish genetic elements.

evolutionary biology↗

bioRxiv2024

NaDyNet: A Toolbox for Dynamic Network Analysis of Naturalistic Stimuli

Experiments with naturalistic stimuli (e.g., listening to stories or watching movies) are emerging paradigms in brain function research. The content of naturalistic stimuli is rich and continuous. The fMRI signals of naturalistic stimuli are complex and include different components. A major challenge is isolate the stimuli-induced signals while simultaneously tracking the brains responses to these stimuli in real-time. To this end, we have developed a user-friendly graphical interface toolbox called NaDyNet (Naturalistic Dynamic Network Toolbox), which integrates existing dynamic brain network analysis methods and their enhanced versions. The main features of NaDyNet are: 1) extracting signals of interest from naturalistic fMRI signals; 2) incorporating six commonly used dynamic analysis methods and three static analysis methods; 3) enhanced versions of these dynamic methods by adopting inter-subject analysis to eliminate the effects of non-interest signals; 4) performing K-means clustering analysis to identify temporally reoccurring states along with their temporal and spatial attributes. We then introduced the rationale for incorporating inter-subject analysis to improve existing dynamic brain network analysis methods, and presented numerous examples. We also summarized research progress in comparing methodological efficacy, offered our recommendations for method selection in dynamic network analysis, and discussed the limitations of current approaches and directions for future research. We hope that this open source toolbox will promote the development of naturalistic neuroscience. The toolbox is available at https://github.com/yuanbinke/Naturalistic-Dynamic-Network-Toolbox.

neuroscience↗

bioRxiv2024

Rab2B promotes chaperonin-mediated actin folding and prevents developmental transcription reprogramming and quiescence in trypanosomes

The Rab family of small GTPases are regulators of intracellular membrane trafficking. Rab2B, a Golgi-resident Rab GTPase, has been shown to function in ER-Golgi transport and autophagosome-lysosome fusion in mammalian cells and Drosophila. In Trypanosoma brucei, the autophagy-related function of Rab2B was conserved and depletion of Rab2B blocked autophagosome-lysosome fusion. Intriguingly, depletion of Rab2B induced differentiation of T. brucei from a proliferative slender form to a quiescent stumpy form, a step crucial for parasite transmission. Chaperonin CCT/TRiC was identified as an effector of Rab2B. Depletion of CCT/TRiC subunits or actin (a known CCT/TRiC substrate), or blocking actin polymerisation with Latrunculin A all triggered differentiation, suggesting a role of Rab2B on CCT/TRiC-mediated actin folding. Using an actin chromobody, we identified a pool of nuclear actin that is crucial for transcription reprogramming and life cycle differentiation in T. brucei. The study revealed a function of Rab2B and actin in T. brucei transcription reprogramming leading to cell differentiation, and an unexpected role of Rab2B and CCT/TRiC mediated actin folding.

cell biology↗

bioRxiv2024

Molecular mechanism of Na+/H+ antiporting in NhaA

Sodium-proton antiporter NhaA of Escherichia coli is a paradigm to investigate the mechanistic basis of the fundamental Na+/H+ exchange in cells. However, all existing crystal structures of NhaA are inward-facing (IF) and the putative outward-facing (OF) structures are still unsolved by experiment, limiting a complete understanding of the transport cycle where Lys300 plays a key role in both structural stability and transport function. Here, we report a set of regular molecular dynamics (MD) simulations that start from the structure predicted by an artificial intelligence method that generates function-relevant alternative conformations. It is found that NhaA rapidly relaxes into either the IF or OF conformation. Further-more, neutralization of Lys300 allows two sodium ions bound to the reaction cavity, which is associated with enhanced conformational sampling. Based on these observations, we propose a sodium-coupled mechanism of Na+/H+ antiporting.

biophysics↗

bioRxiv2024

RetS senses neurotransmitter norepinephrine and adrenergic agents to regulate Pseudomonas aeruginosa virulence

Bacterial cells employ diverse mechanisms to perceive and respond to host-associated molecules, coordinate intracellular signal transduction, and engage in interkingdom communications. In this study, we firstly discovered that the adrenergic antagonist dronedarone (DDR) effectively suppresses quorum sensing (QS) and pathogenesis of the opportunistic pathogen P. aeruginosa. Further analysis revealed that DDR targets RetS, an bacterial surface receptor critical for suppressing the GacS/GacA two-component regulatory system (TCS). Interestingly, the adrenergic agonist norepinephrine (NE) was found to exert opposite effects against RetS, thereby reversing DDR-induced phenotypes in P. aeruginosa. Moreover, we identified the hinge region of RetSDISMED2 serves as a binding pocket of NE and DDR, which provides potential mechanisms of the antagonistic effect of NE and DDR. Our study demonstrated that NE promotes P. aeruginosa infections while DDR can serve as an anti-virulence compound against P. aeruginosa infections. Thus, RetS might serve as a bacterial adrenergic receptor which keeps the NE under constant surveillance. SignificanceSensing and recognition of host-derived molecules, as well as therapeutic drugs, are crucial for the survival of pathogens during infection process. The adrenergic drugs are well known to affect virulence and pathogenesis of the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa). In this work, we have provided in-depth characterization of a Norepinephrine (NE) receptor in P. aeruginosa, the RetS membrane protein. NE is the-adrenergic agonist known as a stress hormone, exerts its activity through interaction with mammalian adrenergic receptors (AR). It has been shown that increased NE concentration during early sepsis can stimulate the growth of various Gram-negative and Gram-positive bacteria, including P. aeruginosa. We revealed that both adrenergic agonist NE and another adrenergic antagonist dronedarone (DDR) are able to interfering the RetS-GacS/GacA signaling cascade of P. aeruginosa and execute opposite impact on the pathogenesis of P. aeruginosa during infections.

microbiology↗

bioRxiv2024

Defining the parameters for sorting of different RNA cargo into Extracellular vesicles

Extracellular vesicles (EVs) are small particles that are released by cells and mediate cell-cell communication by transferring bioactive molecules. RNA cargo of EVs, including coding and non-coding RNAs, can change the behavior of recipient cells, affecting processes like gene expression, proliferation, and apoptosis. Circular RNAs (CircRNA) are a newly appreciated class of regulatory RNAs that are stable, resistant to degradation and have been shown to be enriched in EVs. They play key roles in gene regulation and are also emerging as promising biomarkers for disease diagnosis. While microRNAs (miRNAs) are the most well studied RNA cargo of EVs, very little is known about the mechanisms of enrichment of circRNAs as well as long linear RNAs. Here, we take a comprehensive genome-wide approach to investigate the role of GC%, size, exon count, structuredness and coding potential, in the sorting and enrichment of circular and long linear RNAs into EVs. We found that size and structuredness had a significant role in enriching RNAs into EVs which was consistent across all classes of RNAs. Furthermore, we found that structuredness could explain the relative enrichment of circRNAs over their linear counterparts. These results were validated on existing public databases of circular and linear RNAs in EVs. By identifying and analyzing these factors, we aim to better understand the complex mechanisms behind EV-mediated RNA transfer and its impact on cell communication in both health and disease. This mechanistic understanding of RNA enrichment in EVs is crucial for engineering EVs with selective RNA cargo.

cell biology↗

bioRxiv2024

Impact of oral Chlamydia Vaccination on Host Gut Microbiome and Metabolites Composition

Chlamydia trachomatis, an intracellular pathogen, is recognized as the most common sexually transmitted bacterial infection among women worldwide. Chlamydia infections can lead to undesirable clinical outcomes, including pelvic inflammatory disease and infertility. Recently, the gut has been identified as a niche for Chlamydia colonization; however, despite the biological impact on the host remaining under investigation, oral inoculation of Chlamydia as a whole-organism vaccine has been reported as a promising strategy for preventing genital Chlamydia infections. Few studies have evaluated the impact of oral Chlamydia vaccination on the gut microbiome and metabolite changes. In this study, we assessed time-series alterations in the gut microbiome and metabolites following oral Chlamydia inoculation, and we analyzed the composition and correlation between serum immune parameters and the sequencing profiles in the host. We identified 129 microbial changes and 186 significantly different metabolites in the gut across various vaccination approaches during the 30-day immunization process. Additionally, we discussed potential biomarkers of effective immunization based on correlation analysis. IMPORTANCEChlamydia infections primarily lead to morbidity rather than mortality. Consequently, in developing and implementing a Chlamydia vaccine, the utmost priority is ensuring safety. As a promising yet controversial approach, live oral vaccination for Chlamydia raises concerns regarding its impact on the hosts gut environment. Our study not only investigates changes in the gut microbiome and metabolites during vaccination but also identifies potential biomarkers during immunization. These findings are crucial for the development of whole-organism oral Chlamydia vaccines and provide valuable insights into the long-term colonization of Chlamydia in the gut.

microbiology↗

bioRxiv2024

Ecological response of Angelica dahurica and its rhizosphere microorganisms to treatments of Alternanthera philoxeroides

To explore the ecological adaptation mechanism of the medicinal plant Angelica dahurica and its rhizosphere microorganisms to the invasion of Alternanthera philoxeroides. This study analysed the physiological and biochemical indexes of A. dahurica seedlings and soil enzyme activities under a series concentration of root, stem and leaf extracts from A. philoxeroides. Furthermore, high-throughput sequencing technology was used to detect the rhizosphere microorganism diversity of A. dahurica. Results showed that with the increase of extract concentration and the extension of treatment time, the activity of antioxidant enzymes (SOD, POD, CAT) in seedlings increased and then decreased, and the inhibitory effect of root extract was the most significant. The extract effect on soil enzyme activity followed the descending sequence: root stem leaf. The activities of soil enzymes S-{beta}-GC, S-LAP and S-ALP showed an upward trend with the increase of extract concentration, and the highest enzyme activity was found at 80 g/L. Proteobacteria, Acidobacteria, Bacteroides and Sphingomonas were the dominant species of rhizosphere soil bacteria under the treatment of each extract at phyla level. With the increasing concentration of different organ extracts, the relative abundance of Proteobacteria, Bacteroides and Sphingomonas gradually increased, while the abundance of Acidobacteria decreased. PcoA principal component analysis revealed a significant differentiation between rhizosphere bacterial community structure under the extract and control treatments. These results indicated that different concentrations of A. philoxeroides extract could affect the growth and soil enzyme activity of A. dahurica and change the community structure of rhizosphere microorganisms.

ecology↗

bioRxiv2024

Accurate prediction of CRISPR editing outcomes in somatic cell lines and zygote with few-shot learning

The CRISPR-Cas system has revolutionized gene editing, while its outcome prediction remains unsatisfactory, especially in new cell states due to their distinct DNA repair preferences. In this study, we introduce inDecay, a flexible system for predicting CRISPR editing outcomes from target sequence, returning probabilities of nearly the full spectrum of indel events. Uniquely, inDecay utilizes informative and parameter-efficient features for each indel event and incorporates cell-type-specific repair preferences through a multi-stage design. While both inDecay and existing methods achieve accurate results for prediction within cell lines, only inDecay with transfer learning can retain the high performance for cross-cell line prediction. We then applied inDecay to mouse embryo editing using our newly generated data and observed remarkable accuracy by including as few as 30 fine-tuning embryonic samples. Notably, inDecay is the first software to predict embryonic editing. Therefore, our few-shot learning-supported system may accelerate guide RNA prioritization in mouse model generation, mammal embryonic gene editing, and cellular therapeutics.

bioinformatics↗

bioRxiv2024

Critical Spacing in Cell-Matrix Mechanical Feedback Controls Tissue Condensation

Biological tissues exhibit phase transitions governed by mechanical feedback between cells and their extracellular matrix (ECM). We demonstrate through bio-chemo-mechanical modeling that this emergent behavior arises from competing physical effects: increasing matrix stiffness enhances individual cell activation while simultaneously weakening long-range mechanical communication. This competition establishes a critical cell spacing threshold (80-160 {micro}m) that precisely matches experimental observations across diverse cell types and collagen densities. Our model reveals that the critical stretch ratio at which fibrous networks transition from compliant to strain-stiffening governs this threshold through the formation of tension bands between neighboring cells. These tension bands create a mechanical percolation network that drives the collective phase transition in tissue behavior. Our model explains how fibrous architecture controls emergent mechanical properties in biological systems and offers insight into both the physics of fiber-reinforced composite materials under active stress, and into potential mechanical interventions for fibrotic disorders.

biophysics↗

bioRxiv2024

BMP4 is sufficient to induce a caudal organizer

The caudal part of a vertebrate embryo consists of somites, neural tube, lateral plate mesoderm derivatives and the tailbud. However, the minimal conditions and factors sufficient to induce the caudal region, particularly in humans, remain unresolved. Here, we show that BMP4 alone, when administered at appropriate dosage, is sufficient to induce the formation of an organizer for caudal induction. This organizer induces caudal cell fate specification and morphogenesis in zebrafish embryos. In 3D human pluripotent stem cells (hPSCs) aggregates, BMP4 can induce an elongated embryonic structure characterized by caudal fates. Importantly, hPSCs instructed by BMP4 are sufficient to induce a secondary caudal region when grafted into the animal pole of the zebrafish embryo. Our study thus uncovers BMP4 as the inducer in the formation of a caudal organizer in the vertebrate embryo. Significance statementWe demonstrate that BMP4 alone can induce the formation of a caudal organizer, a critical structure that guides the development of the caudal region in vertebrates. Using zebrafish embryos, embryonic explants, human pluripotent stem cell xenografts, and 3D human stem cell aggregates, we show that this organizer replicates morphogenesis and key differentiation pathways seen in natural development. Our findings uncover a novel role for BMP4 and define the minimal requirements for inducing this organizer, offering new insights into vertebrate development and potential applications in regenerative medicine.

developmental biology↗

bioRxiv2024

PreDigs: a Database of Context-specific Cell-type Markers and Precise cell subtypes for Digestive Cell Annotation

Research on cell type markers aids investigators in exploring the diverse cellular compositions within gastrointestinal tumors, enhancing our understanding of tumor heterogeneity and its implications for disease progression and treatment response. However, issues such as the integration of large-scale datasets and the lack of standardized cell type identification hinder comprehensive characterization. Here, we developed a user-friendly web interface called PreDigs (Predicted Signatures in Digestive System), which offers 124 tailored scRNA-seq datasets available for download, encompassing over 3.4 million cells. After unsupervised clustering, we unified the identification and naming of subtype labels, ultimately constructing a cell ontology tree that includes 142 cell types, with up to eight hierarchical levels. Meanwhile, we calculated three different context-specific cell-type markers--Cell Markers, Subtype Markers, and TPN Markers--based on various application requirements within or across tissues. Through the integrated analysis of PreDigs gastrointestinal data, we identified distinct cell subpopulations exclusive to tumors, one of which corresponds to tumor-specific endothelial cells (TEC). Furthermore, PreDigs offers online cell annotation tools that empower users to perform single-cell classification with greater flexibility, accessible at https://www.biosino.org/predigs/.

bioinformatics↗

bioRxiv2024

AutoPM3: Enhancing Variant Interpretation via LLM-driven PM3 Evidence Extraction from Scientific Literature

Rare diseases, affecting 300 million people globally, often result from genetic variants. Wholegenome sequencing has made variant detection more cost-effective, but interpreting these variants remains challenging. Current clinical practice combines quantitative evidence and literature, which is complex and time-consuming. We introduce AutoPM3, a method for automating the extraction of ACMG/AMP PM3 evidence from scientific literature using open-source LLMs. It combines an optimized RAG system for text comprehension and a TableLLM equipped with Text2SQL for data extraction. We evaluated AutoPM3 using our collected PM3-Bench, a dataset from ClinGen with 1,027 variant-publication pairs. AutoPM3 significantly outperformed other methods in variant hit and in trans variant identification, thanks to the four key modules. Additionally, we wrapped AutoPM3 with a user-friendly interface to enhance its accessibility. This study presents a powerful tool to improve rare disease diagnosis workflows by facilitating PM3-relevant evidence extraction from scientific literature.

bioinformatics↗

bioRxiv2024

Revolutionizing large-scale DNA synthesis with microchip-based massive in parallel synthesis system

DNA synthesis serves as the fundamental enabling technology of engineering biology, aiming to provide DNA molecules of designed composition, length, and complexity at scale and low cost. Current high-throughput DNA synthesis technologies rely on intricate chip manufacturing and microfluidic systems to provide large-scale synthetic oligonucleotides, at the expense of low concentration and limited compatibility in the processing of longer DNA constructs assembly. Here, we report a microchip-based massive in parallel synthesis (mMPS), pioneering an "identification-sorting-synthesis-recycling" iteration mechanism to microchips for high throughput DNA synthesis. In comparison to microarray-based methods, we demonstrate that our method can increase the DNA product concentration by 4 magnitudes (to picomole-scale per sequence) and greatly simplifies the downstream processes for large-scale gene synthesis construction. By the construction of 1.97 million-diversity variant libraries that cover 1,254 human protein domains, we demonstrated the uniformity of the constructed variant libraries using mMPS-derived oligos is greatly improved, with amino acid distribution highly consistent as designed. In addition, by synthesizing 285 1kb-to-3kb genes with varying degrees of sequence complexity from previously reported strains A501 and 3DAC, potential ancestor of early archaea and bacteria, our result shows that the overall gene assembly success rate using mMPS-derived oligos is increased by 10-fold in comparison to other methods. Our mMPS technology holds the potential to close the gap between the quality and cost of writing DNA in increasing demand across many sectors of research and industrial activities.

synthetic biology↗