Biology subjects

Liang, S.

Publications and source records attributed to Liang, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Phosphorylation of the α subunit inhibits proteasome assembly and regulates cell division in archaea

Archaea of the order Sulfolobales possess a eukaryotic-like cell division machinery and display a eukaryotic-like cell cycle, however, the cell division and cell cycle control mechanisms remain enigmatic. Here, we demonstrate that phosphorylation of the subunit by a eukaryotic-like protein kinase ePK2 affects 26S proteasome assembly and controls cell division in Saccharolobus islandicus. ePK2 exhibits cell cycle-dependent expression at both transcriptional and translational levels. Deletion or overexpression of ePK2 results in impaired cytokinesis, with the deletion cells being unable to generate single chromosome cells after synchronization and the overexpression cells exhibiting growth retardation and cell enlargement. Interestingly, overexpression of ePK2 leads to a coherent reduction in cellular proteasome activity and degradation of cell division proteins. We identify S200 and T213 of the proteasome subunit as specific target sites for ePK2 phosphorylation. Functional and structural analyses of site-directed mutants at S200 and T213 suggest that S200 phosphorylation disrupts the assembly of 20S into 26S proteasome whereas T213 phosphorylation interferes the de novo ring assembly. Collectively, our study uncovers an ingenious and efficient mechanism of proteasome phosphorylation-mediated cell division regulation, a prototype of the eukaryotic cell cycle regulation system, in Sulfolobales archaea.

microbiology↗

bioRxiv2025

Unveiling the Developmental Dynamics and Functional Role of Odorant Receptor Co-receptor (Orco) in Aedes albopictus: A Novel Mechanism for Regulating Tuning Odorant Receptor Expression

As one of the most aggressive disease vectors, the Asian tiger mosquito Aedes albopictus relies heavily on its olfactory system to search for food in the larval stage, locate hosts after eclosion, and identify suitable oviposition sites after blood feeding. In mosquitoes and other insects, the olfactory system detects environmental odors primarily through a diverse repertoire of tuning odorant receptors (ORs), which require the highly conserved odorant receptor co-receptor (Orco) to function. While Orcos role in enabling tuning receptor function is well established, its cellular localization patterns, developmental expression dynamics, and system-wide impact on olfactory physiology and behavior remain understudied in Ae. albopictus. To address this knowledge gap, we leveraged the Q-system to systematically characterize Orco-expressing neurons (ORNs) across embryonic, larval, pupal and adult stages of Ae. albopictus. We uncovered a dramatic reorganization of the olfactory system during metamorphosis: ORNs were observed as early as the embryonic stage and expanded during larval development before rapid degeneration and re-generation in the pupal stage resulting in the final population of adult ORNs. Orco expression in adults spanned the olfactory neurons of the antennae, labella, and maxillary palps in both male and female mosquitoes, consistent with its conserved peripheral distribution. To further investigate the functional implications of Orco, we generated Orco knockout mutants and strikingly discovered that Orco knockout mutants displayed significant widespread downregulation of tuning Ors, suggesting that Orco may influence OR expression or stability. Electrophysiological recordings confirmed significantly attenuated responses to human volatiles in Orco mutants, and behavioral assays demonstrated a marked decline in blood-feeding efficiency and elimination of host preference in females. Together, these findings reveal dynamic reorganization of ORNs during mosquito development and uncover the critical role of Orco in maintaining the integrity and function of the olfactory system, providing insights that may inform novel, next-generation vector control strategies.

neuroscience↗

bioRxiv2025

STEAMBOAT: Attention-based multiscale delineation of cellular interactions in tissues

Spatial-omics technologies profile cells in their native spatial context within tissues, enabling more complete understanding of cellular properties. However, a key computational challenge remains: identifying cellular interactions that underlie cell types and states - interactions that are essential for spatial organization and provide a biologically grounded framework for understanding cell identities and spatial patterns. These interactions span different distances and thus require multiscale modeling, which remains a major gap in existing methods. Here, we introduce SO_SCPLOWTEAMBOATC_SCPLOW, an interpretable machine learning framework that leverage a self-supervised, multi-head attention model to uniquely decompose gene expression of a cell into multiple key factors: intrinsic cell programs, neighboring cell communication, and long-range interactions. By applying SO_SCPLOWTEAMBOATC_SCPLOW to diverse tissues in health and disease across various spatial-omics technologies, we demonstrate its ability to uncover critical multiscale cellular interactions, capturing classical contact signaling and revealing previously unrecognized patterns of cellular communication. SO_SCPLOWTEAMBOATC_SCPLOW provides a powerful approach for spatial-omics analysis, offering new insights into the multiscale spatial organization of cells and their communication across a wide range of biological contexts.

bioinformatics↗

bioRxiv2025

The piRNA PETPIR Regulates Trophoblast Function by Modulating YTHDF2-Mediated m6A Methylation of NDRG1 in Preeclampsia

Preeclampsia is marked by abnormal placental development and impaired trophoblast function. We identified Preeclampsia-associated trophoblast piRNA (PETPIR) as an important regulator of trophoblast function and m6A modifications. Placental samples from preeclamptic and healthy pregnancies were analyzed using piRNA microarrays and m6A epitranscriptomic profiling. Functional assays in trophoblast cells, transcriptomic analysis, and an in vivo PETPIR-overexpressing mouse model were employed to investigate PETPIRs mechanistic roles. We found that PETPIR was significantly upregulated in preeclamptic placentas. PETPIR inhibited trophoblast cell proliferation, migration, and invasion by inducing cell cycle arrest and apoptosis. Transcriptome analysis demonstrated that PETPIR activated hypoxia-responsive pathways. Additionally, PETPIR enhanced global m6A methylation and stabilized NDRG1 transcripts by interacting with the m6A reader YTHDF2. In vivo, PETPIR overexpression in pregnant mice recapitulated key features of preeclampsia, including maternal hypertension and fetal growth restriction. These results suggest PETPIR is a key player in the pathogenesis of preeclampsia, functioning through the PETPIR/YTHDF2/NDRG1 axis to drive hypoxia and m6A epitranscriptomic dysregulation. We provide novel insights into preeclampsias molecular mechanisms, highlighting piRNA as a promising biomarker and therapeutic target.

molecular biology↗

bioRxiv2025

A new class of OLD family ATPases functions in cell cycle progression in thermoacidophilic archaea

Overcoming lysogenization defect (OLD) proteins are diverse ATPases-nucleases functioning in antiphage defense in bacteria. However, the role of these proteins in archaea is currently unknown. Here, we describe a new class of archaeal OLD family ATPases and show that they do not appear to be involved in antiviral defense but play an essential role in cell cycle progression. The gene in Saccharolobus islandicus REY15A, named as Cran1 (Cell cycle related ATPase and nickase 1), cannot be deleted and exhibits cyclic expression patterns, with a peak expression during the transition from M-G1 to S cell cycle phase. Cran1 overexpression leads to significant growth retardation, cell size enlargement, and increased cellular DNA content. Cran1 displays potent nickase and ATPase activities in vitro, with the nickase activity being dependent on the presence of the ATPase domain. Notably, Cran1 copurifies with chromatin-associated proteins, such as Cren7 and a histone deacetylase homolog, suggesting its involvement in chromatin-related activities. Collectively our results suggest that Cran1 plays an important role in cell cycle progression, revealing a novel function of OLD family proteins.

microbiology↗

bioRxiv2025

scBiopsy-seq: a platform for temporal single-cell RNA-seq analysis

The development of temporal single-cell RNA-seq (scRNA-seq) assay enabled us to systemically investigate the effects of various types of perturbations in a time course with single-cell resolution. However, the existing temporal scRNA-seq technologies have certain limitations in reliability, detection efficacy and detection diversity. In the current study, we develop scBiopsy-seq assay, which combines a synergistic electroosmosis-electrophoresis extraction method for efficient RNA extraction and digital microfluidics with contamination-isolated hydrophobic interface for high-performance sample processing. scBiopsy-seq extracts the cytoplasm at well-controlled volume to detect >10K genes per extraction with 90% successful rate. Functional enrichment analysis revealed that the genes robustly detected by scBiopsy-seq were associated to diverse biological processes, demonstrating its superb diversity of detection. scBiopsy-seq can perform the sequential extraction of the cytoplasm from the same cell multiple times, which allows us to associate the cell phenotypic responses with its transcriptional dynamics. We employed scBiopsy-seq to analyze the temporal response to a BRD4 degrader induced transcriptional suppression, which identified the key roles of fatty acid beta oxidation in this biological process. scBiopsy-seq with similar data quality as scRNA-seq will dramatically expand the application of temporal scRNA-seq towards a broader spectrum of cell biology research.

bioengineering↗

bioRxiv2025

Multi-omic landscape of human gliomas from diagnosis to treatment and recurrence

Gliomas are among the most lethal cancers, with limited treatment options. To uncover hallmarks of therapeutic escape and tumor microenvironment (TME) evolution, we applied spatial proteomics, transcriptomics, and glycomics to 670 lesions from 310 adult and pediatric patients. Single-cell analysis shows high B7H3+ tumor cell prevalence in glioblastoma (GBM) and pleomorphic xanthoastrocytoma (PXA), while most gliomas, including pediatric cases, express targetable tumor antigens in less than 50% of tumor cells, potentially explaining trial failures. Longitudinal samples of isocitrate dehydrogenase (IDH)-mutant gliomas reveal recurrence driven by tumor-immune spatial reorganization, shifting from T-cell and vasculature-associated myeloid cell-enriched niches to microglia and CD206+ macrophage-dominated tumors. Multi-omic integration identified N-glycosylation as the best classifier of grade, while the immune transcriptome best predicted GBM survival. Provided as a community resource, this study opens new avenues for glioma targeting, classification, outcome prediction, and a baseline of TME composition across all stages.

cancer biology↗

bioRxiv2025

Feature-Driven Whole-Tissue Imaging with Subcellular Resolution

Understanding how subcellular structures and events shape tissue-wide phenomena remains a central challenge in biology. We present Multiscale Cleared Tissue Axially Swept Light-Sheet Microscopy (MCT-ASLM), a platform combining cm-scale imaging with targeted high-resolution interrogation of intact tissues in human-guided or autonomous modes. Capable of capturing fields of view up to 21 mm at micron-scale resolution, MCT-ASLM can seamlessly transition to submicron (~300 nm) isotropic resolution for targeted imaging. This versatility enables comprehensive studies of hierarchical organization and spatially complex processes, including mapping neuronal circuits in rat brains, visualizing glomerular innervation in mouse kidneys, and examining metastatic tumor microenvironments. By bridging subcellular to tissue-level scales, MCT-ASLM offers a powerful method for unraveling how local events contribute to global biological phenomena.

biophysics↗

bioRxiv2025

Evolution of chronic lymphocytic leukemia after allogeneic stem cell transplant

Allogeneic stem cell transplant (alloSCT) for patients with relapsed/refractory chronic lymphocytic leukemia (CLL) can result in cure in some patients. Analogous to chemotherapy and targeted therapy, we hypothesized that allogeneic cellular immunotherapies, including alloSCT and donor lymphocyte infusion (DLI), would impact malignant evolution through the application of selective immunologic pressure with reciprocal changes in the T cell compartment. We tested a cohort of 24 patients treated with HLA-matched alloSCT +/- DLI, two mediators of the graft versus leukemia (GVL) effect. Comparison of pre-alloSCT samples revealed that a key difference between responders (n=13) and non-responders (n=11) is the cellularity of leukemic cells. We further mapped mutational trajectories of tumor cells by whole exome sequencing (WES) of sort-purified CLL in 11 post-transplant relapsed patients and found evidence of subclonal leukemic evolution in 8/11 patients after nonmyeloablative human leukocyte antigen (HLA)-matched alloSCT. Different patterns of CLL evolution were observed, and these changes included putative CLL drivers in every case. To investigate the presence of immune-related variants in patients, we collected 19 T cell co-culture CRISPR datasets and identified the top positive and negative regulators of cancer cells response to T-cell-dependent killing. We found that most mutations linked to T-cell killing emerged after allo-SCT treatment, suggesting that selective pressures from the GVL effect may drive the evolution of these mutations. Together, these data identify cellular homogeneity as a key biomarker for susceptibility to GVL-driven immunity in CLL and illustrate how the leukemic cells further evolve to evade immunosurveillance. SIGNIFICANCEThe impact of allogeneic stem cell transplant (alloSCT) on subclonal leukemia evolution remains poorly understood. By performing whole exome sequencing (WES) on pre- and post-alloSCT patient samples, we reveal different patterns of CLL evolution and potential immune-related driver genes influencing CLL relapse and refractory disease post-alloSCT.

cancer biology↗

bioRxiv2024

HDAC6-Mediated FoxO1 Acetylation And Phosphorylation Control Periodontal Inflammatory Responses

Post-translational modifications (PTMs) are critical regulators of protein function and cellular signaling. While histone deacetylation by histone deacetylases (HDACs) is well established, the role of specific HDACs in modulating non-histone protein PTMs, particularly in an infectious context, is poorly understood. Here, we reveal a pivotal role for HDAC6 in orchestrating periodontal inflammation through its dual regulatory effects on FoxO1 acetylation and phosphorylation. Using Porphyromonas gingivalis, a key periodontal pathogen, as a model pathogen, we observed that infection induces HDAC6 activation, driving inflammatory responses via modulating FoxO1 activity. HDAC6 depletion increased FoxO1 acetylation and phosphorylation, leading to its cytoplasmic sequestration and subsequent suppression of FoxO1- mediated pro-inflammatory cytokine production in macrophages. Mechanistically, HDAC6 deficiency not only directly enhances the acetylation of FoxO1 but also upregulates the expression of Rictor, a critical component of the mTORC2 complex, thereby promoting Akt phosphorylation and subsequently FoxO1 phosphorylation. This results in its cytoplasmic retention and attenuated inflammatory transcriptional activity. Functional studies demonstrated that HDAC6 depletion suppressed the production of key inflammatory mediators, including TNF, IL-6, IL-12p40, and MIP-2, while promoting macrophage polarization toward anti-inflammatory M2 phenotypes. In vivo, using oral gavage infection and ligature-induced mouse periodontitis models, HDAC6 deficiency significantly reduced inflammatory cell infiltration in gingival tissues and protected against alveolar bone loss. These findings establish HDAC6 as a central regulator of periodontal inflammation, acting through the coordinated modulation of FoxO1 acetylation and phosphorylation. Beyond its role in oral pathology, HDAC6 may serve as a promising therapeutic target for managing inflammatory diseases linked to immune dysregulation.

molecular biology↗

bioRxiv2024

BioMedGraphica: An All-in-One Platform for Biomedical Prior Knowledge and Omic Signaling Graph Generation

Multi-omic data analysis is essential for scientific discovery in precision medicine. However, translating statistical results of omic data analysis into novel scientific hypothesis remains a significant challenge. Human experts must manually review analysis results and generate new hypothesis based on extensive and inter-connected biomedical prior knowledge, which is subjective and not scalable. While large language models (LLMs) can accelerate the discovery, their reasoning improves when grounded in structured, auditable and comprehensive biomedical prior knowledge. Biomedical knowledge, however, is scattered across heterogeneous databases that use diverse and inconsistent nomenclature systems, making it difficult to integrate resources into a unified format for scalable analysis. This fragmentation limits the ability of AI systems to fully leverage biomedical data for scientific discovery. To address these challenges, we developed BioMedGraphica, an all-in-one platform that harmonizes fragmented biomedical resources by integrating 11 entity types and 30 relation types from 43 databases into a unified knowledge graph containing 2,306,921 entities and 27,232,091 relations. In addition, to the best of our knowledge, this is the first work to propose a novel Textual-Numeric Graph (TNG) data-structure for multi-omics data analysis. In TNG, textual information captures prior biological knowledge (e.g., transcription start sites, functions, mechanisms), while numeric values represent quantitative biomedical features, and the integrated relations can help uncover mechanisms. By bridging prior knowledge with user-specific data, TNG is a novel and ideal data-structure for the development of graph foundation models, with the potential to improve prediction performance and interpretability, while also augmenting LLMs by supplying graph-structured mechanistic context to strengthen reasoning. The details for BioMedGraphica code can be accessed by github link: https://github.com/FuhaiLiAiLab/BioMedGraphica and BioMedGraphica knowledge graph data can be downloaded from huggingface dataset: https://huggingface.co/datasets/FuhaiLiAiLab/BioMedGraphica

bioinformatics↗

bioRxiv2024

Population-based metaproteomics reveals functional associations between gut microbiota and phenotypes

The association between gut microbiota and physiological disorders underscores the crucial need to understand its largely unexplored biological functions. In this study, we analyzed 86,876 microbial proteins and 1,277 human proteins from 1,967 Chinese stool samples. Our results indicate that metaproteomic data captures the microbial biodiversity of a population more effectively than metagenomic data, displaying greater variability in microbial functions and providing reliable assessments of microbiota stability. We identified crucial functions of core microbes and discovered 10,714 associations between metaproteomic taxon, microbial function, or human proteins with 39 phenotypes, including 1,381 features related to Type 2 Diabetes (T2D). Importantly, Megasphaera elsdeniis conversion of lactate to butyric acid appears to lower blood glucose levels, suggesting a protective mechanism against T2D. Our study underscores the power of population-based metaproteomics to offer unique functional insights into the gut microbiota. Highlights- The first large-scale population-based metaproteomics landscape. - Metaproteomics efficiently maps microbial functions, exposing individual functional variation. - 10,714 associations between metaproteomic taxa, microbial functions, or human proteins with 39 phenotypes. - Megasphaera elsdenii protects against elevated blood glucose levels in the host by degrading lactate to produce butyric acid.

microbiology↗

bioRxiv2024

Integration of hunger and hormonal state gates infant-directed aggression

Social behaviour is profoundly shaped by internal physiological states. While significant progress has been made in understanding how individual states such as hunger, stress, or arousal modulate behaviour, animals experience multiple states at any given time. The neural mechanisms that integrate such orthogonal states--and how this integration affects behaviour--remain poorly understood. Here we report how hunger and estrous state converge on neurons in the medial preoptic area (MPOA) to shape infant-directed behaviour. We find that hunger promotes pup-directed aggression in normally non-aggressive virgin female mice. This behavioural switch occurs through inhibition of MPOA neurons, driven by the release of neuropeptide Y (NPY) from Agouti-related peptide-expressing neurons in the arcuate nucleus (ArcAgRP neurons). The propensity for hunger-induced aggression is set by reproductive state, with MPOA neurons detecting changes in progesterone (P4) to estradiol (E2) ratio across the estrous cycle. Hunger and estrous state converge on HCN (hyperpolarization-activated cyclic nucleotide-gated) channels, which sets the baseline activity and excitability of MPOA neurons. Using micro-endoscopic imaging, we confirm these findings in vivo, revealing that MPOA neurons encode a state for pup-directed aggression. This work thus provides a mechanistic understanding of how multiple physiological states are integrated to flexibly control social behaviour.

neuroscience↗

bioRxiv2024

A Novel C. elegans Model for Tau spreading Reveals Genes Critical for Endolysosomal Integrity and Seeded Tau Aggregation

The spreading of Tau pathology is closely associated with the progression of neurodegeneration and cognitive decline in Alzheimers disease and other tauopathies. A key event in this process is the rupture of endolysosomal vesicles following the intercellular transfer of Tau aggregates, releasing the transferred Tau species into the cytosol where they can promote the aggregation of endogenous Tau. However, understanding of the cellular pathways involved in this process remains limited. In this study, we investigated cellular pathways that prevent endolysosomal vesicle rupture. We established a new C. elegans model of Tau spreading by introducing an mCherry-labelled, disease-associated aggregation-prone fragment of human Tau (F3{Delta}K281::mCh) into the six touch receptor neurons. F3{Delta}K281::mCh transgenic animals exhibited significant neurotoxicity and mechanosensory deficits due to the accumulation of this Tau fragment. In addition, its intercellular transmission compromised the endolysosomal system in receiving hypodermal cells. Using this model, we conducted an unbiased genome-wide RNAi screen and identified 59 genes critical for maintaining endolysosomal integrity. GO-term analysis revealed an enrichment of genes related to the ESCRT complex, the ubiquitin-proteasome system, mRNA splicing, and fatty acid metabolism. Silencing of selected conserved genes exacerbated seeded Tau aggregation in a human induced pluripotent stem cell (hiPSC)-derived cortical neuron model and triggered endolysosomal rupture in HEK293T cells, confirming the crucial role of endolysosomal damage in seeded Tau aggregation. Overall, this study discovered novel cellular pathways that safeguard endolysosomal integrity. These findings may guide the development of therapeutics that improve endolysosomal integrity to halt the progression of Tau pathology.

cell biology↗

bioRxiv2024

Brainstem neurons coordinate the bladder and urethra sphincter for urination

Urination, a vital and conserved process of emptying urine from the urinary bladder in mammals, requires precise coordination between the bladder and external urethral sphincter (EUS) that is tightly controlled by a complex neural network. However, the specific subpopulation of neurons that accounts for such coordination remains unidentified, limiting the development of target-specific therapies for certain urination disorders, e.g., detrusor-sphincter dyssynergia. Here, we find that cells expressing estrogen receptor 1 (ESR1+) in the pontine micturition center (PMC) initiate voiding when activated and suspend ongoing voiding when suppressed, each at 100% reliability. Transection of the pelvic nerve does not impair PMCESR1+ neurons control of the EUS via the pudendal nerve, whereas transection of the pudendal nerve does not impair their control of the bladder via the pelvic nerve. Anatomically, PMCESR1+ neurons consist of three distinct spinal-projection-based subpopulations: one targeting the sacral parasympathetic nucleus (SPN), one innervating the dorsal gray commissure (DGC), and a third that projects to both regions, thereby enforcing the coordination of bladder contraction and sphincter relaxation in a rigid temporal sequence. Thus, we identify a cell type in the brainstem that controls the bladder-urethra coordination for urination.

neuroscience↗

bioRxiv2024

Iterative sacrificial 3D printing and polymer casting to create complex vascular grafts and multi-compartment bioartificial organs

Several emerging strategies to engineer artificial organs employ 3D printing to create vascular templates to provide nutrients and oxygen to immobilized cells. Significant challenges emerge when considering clinical implementation such as immune rejection of allogeneic cell sources, as well as achieving adequate perfusion and integration with endogenous vasculature. We propose a method by which cell-laden hydrogels are molded around ready-made polymeric vascular templates created via 3D printing to create human-scale artificial organs with internal vasculature. We applied this technique to create bioartificial pancreas systems with up to 9 internal flow channels via sacrificial carbohydrate glass 3D printing, porogen-loaded polycarbonate polyurethane dip-coating, followed by casting cell-laden hydrogels around the vascular templates. We optimized porogen size and concentration to maximise the porosity of our scaffolds without compromising mechanical properties, resulting in suture retention strength and compliance respectively matching commercial vascular grafts and native vessels. Bioreactor perfusion studies showed survival and maturation of stem cell derived pancreatic islets without significant differences to traditional suspension culture protocols. Insulin response dynamics were rapid in response to a glucose challenge at the perfusion inlet. Transplantation of the devices as iliac arteriovenous shunts in nondiabetic pigs confirmed safety and patency. These results show promise for the development of an implantable vascularized pancreas for the treatment of type 1 diabetes and demonstrate how bioartificial organs with engineered vascular geometries can be designed for translational applications.

bioengineering↗

bioRxiv2024

Identification of novel components of the Ced and Ups systems in Saccharolobus islandicus REY15A

In the cells of Sulfolobales, the Ced (Crenarchaeal system for exchange of DNA) and Ups (UV-inducible pili of Sulfolobus) systems are highly induced by DNA damages and function in chromosomal DNA exchange by mediating DNA import and cell aggregation, respectively. The Ced system is composed of CedA, CedA1, CedA2, and CedB. The Ups is composed of UpsA, UpsB, UpsE and UpsF. However, how the DNA is transported by these systems is far from clear. Here, we report two novel components of the Ced system, CedD (SiRe_1715) and CedE (SiRe_2100), which are paralogs of CedB and CedA, respectively, and one new component of the Ups system, UpsC (SiRe_1957), which is a paralog of UpsA and UpsB, in Saccharolobus islandicus REY15A. DNA exchange assay revealed that cedD, cedE, and upsC are essential for DNA import while cedE and upsC are also important for DNA export. Further microscopy analysis revealed that upsC is involved in cell aggregation like other Ups genes. Besides, we found that cedB and cedD co-occur in the genomes of crenarchaea which lack virB4 and the presence of cedB and cedD, and virB4 is complementary. Both proteins show homology to different parts of the N-terminal domain of VirB4 in a complementary manner and form stable homo-oligomers in vitro. Collectively, our results indicate that CedD and CedE, and UpsC are essential components of the Ced and Ups systems, respectively.

microbiology↗

bioRxiv2024

Signal sequences target enzymes and structural proteins to bacterial microcompartments and are critical for microcompartment formation

Spatial organization of pathway enzymes has emerged as a promising tool to address several challenges in metabolic engineering, such as flux imbalances and off-target product formation. Bacterial microcompartments (MCPs) are a spatial organization strategy used natively by many bacteria to encapsulate metabolic pathways that produce toxic, volatile intermediates. Several recent studies have focused on engineering MCPs to encapsulate heterologous pathways of interest, but how this engineering affects MCP assembly and function is poorly understood. In this study, we investigated the role of signal sequences, short domains that target proteins to the MCP core, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterized two novel Pdu signal sequences on the structural proteins PduM and PduB, which constitutes the first report of metabolosome signal sequences on structural proteins rather than enzymes. We then explored the role of enzymatic and structural Pdu signal sequences on MCP assembly by deleting their encoding sequences from the genome alone and in combination. Deleting enzymatic signal sequences decreased MCP formation, but this defect could be recovered in some cases by overexpressing genes encoding the knocked-out signal sequence fused to a heterologous protein. By contrast, deleting structural signal sequences caused similar defects to knocking out the genes encoding the full length PduM and PduB proteins. Our results contribute to a growing understanding of how MCPs form and function in bacteria and provide strategies to mitigate assembly disruption when encapsulating heterologous pathways in MCPs.

synthetic biology↗