Biology subjects

Jung, S.

Publications and source records attributed to Jung, S..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

A distinct CD115- erythro-myeloid precursor present at the maternal-embryonic interface and in the bone marrow of adult mice

During ontogeny, macrophages develop from CD115+ precursors, including erythro-myeloid progenitors (EMP). EMP arise in the embryonic yolk sac, the primary site of early haematopoiesis. In adults, CD115+ bone marrow-derived monocytes represent essential macrophage precursors. Herein, we identify a CD115- macrophage precursor within the adult bone marrow that is unrelated to the classical monocyte lineage but rather shares transcriptomic and functional characteristics of embryonic EMP. These EMPROR (for Erythro Myeloid Precursor) cells are capable of efficiently generating macrophages in disease settings. During early development, EMPROR cells were largely absent from the yolk sac but were instead found at the embryonic-maternal interface in the uterine wall. Unexpectedly, the latter site contains robust haematopoietic activity and harbours defined embryonic haematopoietic progenitor cells, including classical CD115+ EMP. Our data suggest the existence of an alternative pathway of macrophage generation in the adult. Further, we uncover a hitherto unknown site of earliest blood cell development.

immunology↗

bioRxiv2021

Structure and lipid-mediated remodelling mechanism of the Na+/H+ exchanger NHA2

Na+/H+ exchangers catalyse an ion-exchange activity that is carried out in most, if not all cells. SLC9B2, also known as NHA2, correlates with the long-sought after sodium/lithium (Na+/Li+) exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite its functional importance, structural information and the molecular basis of its ion-exchange mechanism have been lacking. Here, we report the cryo EM structures of bison NHA2 in detergent and in nanodiscs at 3.0 and 3.5 [A] resolution, respectively. NHA2 shares closest structural similarity to the bacterial electrogenic Na+/H+ antiporter NapA, rather than other mammalian SLC9A members. Nevertheless, SSM-based electrophysiology results with NHA2 show the catalysis of electroneutral rather than electrogenic ion exchange, and the ion-binding site is quite distinctive, with a tryptophan-arginine- glutamate triad separated from the well-established ion-binding aspartates. These triad residues fine-tune ion binding specificity, as demonstrated by a salt-bridge swap mutant that converts NHA2 into a Li+-specific transporter. Strikingly, an additional N-terminal helix in NHA2 establishes a unique homodimer with a large [~] 25 [A] intracellular gap between protomers. In the presence of phosphatidylinositol lipids, the N-terminal helix rearranges and closes this gap. We confirm that dimerization of NHA2 is required for activity in vivo, and propose that the N- terminal helix has evolved as a lipid-mediated remodelling switch for regulation of transport activity.

biophysics↗

bioRxiv2021

SARS-Cov-2 Spike binding to ACE2 is stronger and longer ranged with glycans

Highly detailed steered Molecular Dynamics simulations are performed on differently glycosylated receptor binding domains of the SARS-CoV-2 spike protein. The binding strength and the binding range increases with glycosylation. The interaction energy rises very quickly with pulling the proteins apart and only slowly drops at larger distances. We see a catch slip type behavior where interactions during pulling break and are taken over by new interactions forming. The dominant interaction mode are hydrogen bonds but Lennard-Jones and electrostatic interactions are relevant as well. Statement of SignificanceGlycosylation of the receptor binding domain of the Spike protein of SARS-CoV-2 as well as the ACE2 receptor leads to stronger and longer ranged binding interactions between the proteins. Particularly, at shorter distances the interactions are between residues of the proteins themselves whereas at larger distances these interactions are mediated by the glycans. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/452507v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@7b9021org.highwire.dtl.DTLVardef@338aadorg.highwire.dtl.DTLVardef@1d5921eorg.highwire.dtl.DTLVardef@6c27e7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Enteric glial cells favour accumulation of anti-inflammatory macrophages during the resolution of muscularis inflammation

ObjectiveMonocyte-derived macrophages (M{varphi}s) are crucial regulators during muscularis inflammation. However, it is unclear which microenvironmental factors are responsible for monocyte recruitment and neurotrophic M{varphi} differentiation in this paradigm. Here, we investigate M{varphi} heterogeneity at different stages of muscularis inflammation and determine how environmental cues can attract and activate tissue protective M{varphi}s. DesignSingle cell RNA sequencing was performed on immune cells from the muscularis of wild-type and CCR2-/- mice at different timepoints after muscularis inflammation. CX3CR1GFP/+ and CX3CR1CreERT2 R26YFP mice were analyzed by flow cytometry and immunofluorescence. The transcriptome of enteric glial cells (EGCs) was investigated using PLPCreERT2 Rpl22HA mice. In addition, we assessed the effect of supernatant from neurosphere-derived EGCs on monocyte differentiation based on the expression of pro- and anti-inflammatory factors. ResultsMuscularis inflammation induced marked alterations in mononuclear phagocyte populations associated with a rapid infiltration of Ly6c+ monocytes that locally acquired unique transcriptional states. Trajectory inference analysis revealed two main pro-resolving M{varphi} subpopulations during the resolution of muscularis inflammation, i.e. Cd206+ MhcIIhi and Timp2+ MhcIIlo M{varphi}s, which were both derived from CCR2+ monocytes. Interestingly, we found that EGCs were able to sense damage to the muscularis to stimulate monocyte recruitment and differentiation towards pro-resolving M{varphi}s via CCL2 and CSF1, respectively. ConclusionOur study provides a comprehensive insight into pro-resolving M{varphi} differentiation and their regulators during muscularis inflammation. We deepened our understanding in the interaction between EGCs and M{varphi}s, thereby highlighting pro-resolving M{varphi} differentiation as a potential novel therapeutic strategy for the treatment of intestinal inflammation.

immunology↗

bioRxiv2021

Ligands binding to the cellular prion protein induce its protective proteolytic release with therapeutic potential in neurodegenerative proteinopathies

The cellular prion protein (PrPC) is a central player in neurodegenerative diseases caused by protein misfolding, such as prion diseases or Alzheimers disease (AD). Expression levels of this GPI-anchored glycoprotein, especially at the neuronal cell surface, critically correlate with various pathomechanistic aspects underlying these diseases, such as templated misfolding (in prion diseases) and neurotoxicity and, hence, with disease progression and severity. In stark contrast to cell-associated PrPC, soluble extracellular forms or fragments of PrP are linked with neuroprotective effects, which is likely due to their ability to interfere with neurotoxic disease-associated protein conformers in the interstitial fluid. Fittingly, the endogenous proteolytic release of PrPC by the metalloprotease ADAM10 ( shedding) was characterized as a protective mechanism. Here, using a recently generated cleavage-site specific antibody, we shed new light on earlier studies by demonstrating that shed PrP (sPrP) negatively correlates with conformational conversion (in prion disease) and is markedly redistributed in murine brain in the presence of prion deposits or AD-associated amyloid plaques indicating a blocking and sequestrating activity. Importantly, we reveal that administration of certain PrP-directed antibodies and other ligands results in increased PrP shedding in cells and organotypic brain slice cultures. We also provide mechanistic and structural insight into this shedding-stimulating effect. In addition, we identified a striking exception to this, as one particular neuroprotective antibody, due to its special binding characteristics, did not cause increased shedding but rather strong surface clustering followed by fast endocytosis and degradation of PrPC. Both mechanisms may contribute to the beneficial action described for some PrP-directed antibodies/ligands and pave the way for new therapeutic strategies against devastating and currently incurable neurodegenerative diseases.

neuroscience↗

bioRxiv2021

Disease-specific eQTL screening reveals an anti-fibrotic effect of AGXT2 in nonalcoholic fatty liver disease

Background & AimsNonalcoholic fatty liver disease (NAFLD) poses an impending clinical burden. Genome-wide association studies have revealed a limited contribution of genomic variants to the disease, requiring alternative but robust approaches to identify disease-associated variants and genes. We carried out a disease-specific expression quantitative trait loci (eQTL) screen to identify novel genetic factors that specifically act on NAFLD progression on the basis of genotype. MethodsWe recruited 125 Korean biopsy-proven NAFLD patients and healthy individuals and performed eQTL analyses using 21,272 transcripts and 3,234,941 genotyped and imputed SNPs. We then selected eQTLs that were detected only in the NAFLD group, but not in the control group (i.e., NAFLD-eQTLs). An additional cohort of 162 Korean NAFLD individuals was used for replication. The function of the selected eQTL toward NAFLD development was validated using HepG2, primary hepatocytes and NAFLD mouse models. ResultsThe NAFLD-specific eQTL screening yielded 242 loci. Among them, AGXT2, encoding alanine-glyoxylate aminotransferase 2, displayed decreased expression in NAFLD patients homozygous for the non-reference allele of rs2291702, compared to no-NAFLD subjects with the same genotype (P = 4.79 x 10-6). This change was replicated in an additional 162 individuals, yielding a combined P-value of 8.05 x 10-8 from a total of 245 NAFLD patients and 48 controls. Knockdown of AGXT2 induced palmitate-overloaded hepatocyte death by increasing ER stress, and exacerbated NAFLD diet-induced liver fibrosis in mice. However, overexpression of AGXT2 reversely attenuated liver fibrosis and steatosis as well. ConclusionsWe implicate a new molecular role of AGXT2 in NAFLD. Our overall approach will serve as an efficient tool for uncovering novel genetic factors that contribute to liver steatosis and fibrosis in patients with NAFLD. Lay summaryElucidating causal genes for NAFLD has been challenging due to limited tissue availability and the polygenic nature of the disease. Using liver and blood samples from 125 biopsy-proven NAFLD and no-NAFLD Korean individuals and an additional 162 individuals for replication, we devised a new analytic method to identify causal genes. Among the candidates, we found that AGXT2-rs2291702 protects against liver fibrosis in a genotype-dependent manner with the potential for therapeutic interventions. Our approach enables the discovery of NAFLD causal genes that act on the basis of genotype.

genetics↗

bioRxiv2021

Svep1 stabilizes developmental vascular anastomosis in reduced flow conditions.

We report the discovery that flow and Svep1 are modulator of vessel anastomosis during developmental angiogenesis in zebrafish embryos. We show that loss of Svep1 and blood flow reduction both contribute to defective anastomosis of intersegmental vessels. We show that this defect in primary angiogenic sprouts is associated with an expansion of Apelin-positive tip cells and with reduced formation and lumenisation of the dorsal longitudinal anastomotic vessel. Mechanistically, our results suggest that flow and Svep1 act synergistically to modulate vascular network formation in the zebrafish trunk.

developmental biology↗

bioRxiv2021

Transcriptome profiling of human pluripotent stem cell-derived cerebellar organoids reveals fastercommitment under dynamic conditions

Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling. However, generating iPSC-derived models to study brain diseases remains a challenge. In particular, the ability to recapitulate cerebellar development in vitro is still limited. We presented a reproducible and scalable production of cerebellar organoids by using the novel Vertical-Wheel single-use bioreactors, in which functional cerebellar neurons were obtained. Here, we evaluate the global gene expression profiles by RNA sequencing (RNA-seq) across cerebellar differentiation, demonstrating a faster cerebellar commitment in this novel dynamic differentiation protocol. Furthermore, transcriptomic profiles suggest a significant enrichment of extracellular matrix (ECM) in dynamic-derived cerebellar organoids, which can better mimic the neural microenvironment and support a consistent neuronal network. Thus, an efficient generation of organoids with cerebellar identity was achieved for the first time in a continuous process using a dynamic system without the need of organoids encapsulation in ECM-based hydrogels, allowing the possibility of large-scale production and application in high-throughput processes. The presence of factors that favors angiogenesis onset was also detected in dynamic condition, which can enhance functional maturation of cerebellar organoids. We anticipate that large-scale production of cerebellar organoids may help developing models for drug screening, toxicological tests and studying pathological pathways involved in cerebellar degeneration.

bioengineering↗

bioRxiv2021

Real-Time Three-Dimensional Tracking of Single Vesicles Reveals the Abnormal Motion and Vesicle Pools of Synaptic Vesicles in Neurons of Huntington's Disease Mice

Although defective synaptic transmission was suggested to play a role in neurodegenerative diseases, the dynamics and vesicle pools of synaptic vesicles during neurodegeneration remain elusive. Here, we performed real-time three-dimensional tracking of single synaptic vesicles in cortical neurons from a mouse model of Huntingtons disease (HD). Vesicles in HD neurons had a larger net displacement and radius of gyration compared with wild-type neurons. Vesicles with a high release probability (Pr) were interspersed with low-Pr vesicles in HD neurons, whereas high-Pr and low-Pr vesicle pools were spatially separated in wild-type neurons. Non-releasing vesicles in HD neurons had an abnormally high prevalence of irregular oscillatory motion. These abnormal dynamics and vesicle pools were rescued by overexpressing Rab11, and the abnormal irregular motion was rescued by jasplakinolide. These results suggest the abnormal dynamics and vesicle pools of synaptic vesicles in the early stages of HD, suggesting a possible pathogenic mechanism of neurodegenerative diseases.

neuroscience↗

bioRxiv2021

Homogenization of a Reaction Diffusion Equation can Explain Influenza A Virus Load Data

We study the influence of spatial heterogeneity on the antiviral activity of mouse embryonic fibroblasts (MEF) infected with influenza A. MEF of type Ube1L-/- are composed of two distinct sub-populations, the strong type that sustains a strong viral infection and the weak type, sustaining a weak viral load. We present new data on the virus load infection of Ube1L-/-, which have been micro-printed in a checker board pattern of different sizes of the inner squares. Surprisingly, the total viral load at one day after inoculation significantly depends on the sizes of the inner squares. We explain this observation by using a reaction diffusion model and we show that mathematical homogenization can explain the observed inhomogeneities. If the individual patches are large, then the growth rate and the carrying capacity will be the arithmetic means of the patches. For finer and finer patches the average growth rate is still the arithmetic mean, however, the carrying capacity uses the harmonic mean. While fitting the PDE to the experimental data, we also predict that a discrepancy in virus load would be unobservable after only half a day. Furthermore, we predict the viral load in different inner squares that had not been measured in our experiment and the travelling distance the virions can reach after one day.

microbiology↗

bioRxiv2020

Ribonucleotide reductase, a novel target for gonorrhea

Antibiotic resistant Neisseria gonorrhoeae (Ng) are an emerging public health threat due to increasing numbers of multidrug resistant (MDR) organisms. We identified two novel orally active inhibitors, PTC-847 and PTC-672, that exhibit a narrow spectrum of activity against Ng including MDR isolates. By selecting organisms resistant to the novel inhibitors and sequencing their genomes, we identified a new therapeutic target, the class Ia ribonucleotide reductase (RNR). Activity studies and negative stain electron microscopy of the Ng Ia RNR suggest that these inhibitors potentiate conversion of its active 2{beta}2 state to an inactive 4{beta}4 similar to states first identified with the Escherichia coli (Ec) Ia RNR. Resistance mutations in Ng map to the N-terminal, ATP cone domain of its subunit and disrupt the interaction with the {beta} subunit required to form the specific quaternary inhibited state. Oral administration of PTC-672 reduces Ng infection in a mouse model and may have therapeutic potential for treatment of Ng that is resistant to current drugs.

microbiology↗

bioRxiv2020

In-vivo histology of iron and myelin in the brain using magnetic susceptibility source separation in MRI

Obtaining a histological fingerprint from the in-vivo brain has been a long-standing target of magnetic resonance imaging (MRI). In particular, non-invasive imaging of iron and myelin, which are involved in normal brain functions and are histopathological hallmarks in neurodegenerative diseases, has practical utilities in neuroscience and medicine. Here, we propose a biophysical model that describes the individual contribution of paramagnetic (e.g., iron) and diamagnetic (e.g., myelin) susceptibility sources to the frequency shift and transverse relaxation of MRI signals. Using this model, we develop a method,{chi} -separation, that generates the voxel-wise distributions of the two sources. The method is validated using computer simulation and phantom experiments, and applied to ex-vivo and in-vivo brains. The results delineate the well-known histological features of iron and myelin in the specimen, healthy volunteers, and multiple sclerosis patients. This new technology may serve as a practical tool for exploring the microstructural information of the brain.

neuroscience↗

bioRxiv2020

Neural inflammation alters synaptic plasticity probed by 10 Hz repetitive magnetic stimulation

Systemic inflammation is associated with alterations in complex brain functions such as learning and memory. However, diagnostic approaches to functionally assess and quantify inflammation-associated alterations in synaptic plasticity are not well-established. In previous work, we demonstrated that bacterial lipopolysaccharide (LPS)-induced systemic inflammation alters the ability of hippocampal neurons to express synaptic plasticity, i.e., the long-term potentiation (LTP) of excitatory neurotransmission. Here, we tested whether synaptic plasticity induced by repetitive magnetic stimulation (rMS), a non-invasive brain stimulation technique used in clinical practice, is affected by LPS-induced inflammation. Specifically, we explored brain tissue cultures to learn more about the direct effects of LPS on neural tissue, and we tested for the plasticity-restoring effects of the anti-inflammatory cytokine interleukin 10 (IL10). As shown previously, 10 Hz repetitive magnetic stimulation (rMS) of organotypic entorhino-hippocampal tissue cultures induced a robust increase in excitatory neurotransmission onto CA1 pyramidal neurons. Furthermore, LPS-treated tissue cultures did not express rMS-induced synaptic plasticity. Live-cell microscopy in tissue cultures prepared from a novel transgenic reporter mouse line [C57BL6-Tg(TNFa-eGFP)] confirms that ex vivo LPS administration triggers microglial tumor necrosis factor alpha (TNF) expression, which is ameliorated in the presence of IL10. Consistent with this observation, IL10 hampers the LPS-induced increase in TNF, IL6, IL1{beta}, and IFN{gamma} and restores the ability of neurons to express rMS-induced synaptic plasticity in the presence of LPS. These findings establish organotypic tissue cultures as a suitable model for studying inflammation-induced alterations in synaptic plasticity, thus providing a biological basis for the diagnostic use of transcranial magnetic stimulation in the context of brain inflammation.

neuroscience↗

bioRxiv2020

Unique dynamics and exocytosis of single inhibitory synaptic vesicles

Maintaining the balance between neuronal excitation and inhibition is essential for proper function of the central nervous system, with inhibitory synaptic transmission playing an important role. Although inhibitory transmission has higher kinetic demands compared to excitatory transmission, its properties are poorly understood. In particular, the dynamics and exocytosis of single inhibitory vesicles have not been investigated, due largely to both technical and practical limitations. Using a combination of quantum dots (QDs) conjugated to antibodies against the luminal domain of the vesicular GABA transporter (VGAT) to selectively label GABAergic (i.e., inhibitory) vesicles together with dual-focus imaging optics, we tracked the real-time three-dimensional position of single inhibitory vesicles up to the moment of exocytosis (i.e., fusion). Using three-dimensional trajectories, we found that inhibitory synaptic vesicles traveled a short distance prior to fusion and had a shorter time to fusion compared to synaptotagmin-1 (Syt1)-labeled vesicles, which were mostly from excitatory neurons. Moreover, our analysis revealed a close correlation between the release probability of inhibitory vesicles and both the proximity to their fusion site and the total travel length. Finally, we found that inhibitory vesicles have a higher prevalence of kiss-and-run fusion compared than Syt1-labeled vesicles. These results indicate that inhibitory synaptic vesicles have a unique set of dynamics and fusion properties to support rapid synaptic inhibition, thereby maintaining a tightly regulated balance between excitation and inhibition in the central nervous system. SignificanceDespite playing an important role in maintaining brain function, the dynamics of inhibitory synaptic vesicles are poorly understood. Here, we tracked the three-dimensional position of single inhibitory vesicles up to the moment of exocytosis in real time by loading single inhibitory vesicle with QDs-conjugated to antibodies against the luminal domain of the vesicular GABA transporter (VGAT). We found that inhibitory synaptic vesicles have a smaller total travel length before fusion, a shorter fusion time, and a higher prevalence of kiss-and-run than synaptotagmin-1-lableled vesicles. Our findings provide the first evidence that inhibitory vesicles have a unique set of dynamics and exocytosis properties to support rapid inhibitory synaptic transmission.

neuroscience↗

bioRxiv2020

Plant NLR targets P-type ATPase for executing plasma membrane depolarization leading to calcium influx and cell death

Hypersensitive response (HR) is a robust immune response mediated by plant nucleotide-binding and leucine-rich repeat receptor (NLR). However, the early molecular event linking NLR to cell death is obscure. Here we demonstrate that NLR targets plasma membrane H+-ATPases (PMA) generating electrochemical potential across the membrane. CCA309, an autoactive N-terminal domain of pepper coiled-coil NLR (CNL), associates with PMAs and its autoactivity is affected by silencing or overexpression of PMA. CCA309-induced extracellular alkalization accompanied with membrane depolarization is followed by calcium influx and cell death. CCA309 interacts with C-terminal regulatory domain of PMA and 14-3-3 negatively affects CCA309-induced cell death. Moreover, pharmacological experiments with fusicoccin, an irreversible PMA activator, confirmed that CC- and CNL-mediated cell death occurred through inhibiting PMA. We propose PMAs as the primary target of plasma membrane-associated CNL to disrupt electrochemical homeostasis leading to HR cell death.

plant biology↗

bioRxiv2020

Ermin deficiency as an inside-out model of inflammatory dysmyelination

Ermin is an actin-binding protein found almost exclusively in the central nervous system (CNS) as a component of myelin sheaths. Although Ermin has been predicted to play a role in the formation and stability of myelin sheaths, this has not been directly examined in vivo. Here we show that Ermin is essential for myelin sheath integrity and normal saltatory conduction. Loss of Ermin in mice caused de-compacted and fragmented myelin sheaths and led to slower conduction along with progressive neurological deficits. RNA sequencing of the corpus callosum, the largest white matter structure in the CNS, pointed to inflammatory activation in aged Ermin-deficient mice, which was corroborated by increased levels of microgliosis and astrogliosis. The inflammatory milieu and myelin abnormalities were further associated with increased susceptibility to immune-mediated demyelination insult in Ermin knockout mice. Supporting a possible role of Ermin deficiency in inflammatory white matter disorders, a rare inactivating mutation in the ERMN gene was identified in multiple sclerosis patients. Our findings demonstrate a critical role for Ermin in maintaining myelin integrity. Given its near exclusive expression in myelinating oligodendrocytes, Ermin deficiency represents a compelling "inside-out" model of inflammatory dysmyelination and may offer a new paradigm for the development of myelin stability-targeted therapies.

neuroscience↗

bioRxiv2020

Vasohibin-1 mediated tubulin detyrosination selectively regulates secondary sprouting and lymphangiogenesis in the zebrafish trunk

Previous studies have shown that Vasohibin-1 (Vash-1) is stimulated by VEGFs in endothelial cells and that its overexpression interferes with angiogenesis in vivo. Recently, Vasohibin-1 was found to mediate tubulin detyrosination, a post-translational modification that is implicated in many cell functions, such as cell division. Here we used the zebrafish embryo to investigate the cellular and subcellular mechanisms of Vash-1 on endothelial microtubules during formation of the trunk vasculature. We show that microtubules within venous-derived secondary sprouts are strongly and selectively detyrosinated in comparison with other endothelial cells, and that this difference is lost upon vash-1 knockdown. Vasohibin-1 depletion in zebrafish specifically affected secondary sprouting from the posterior cardinal vein, increasing both the number of sprouts and endothelial cell divisions. We show that altering secondary sprout numbers and structure upon vash-1 depletion leads to a failure in the development and specification of lymphatic vessels of the zebrafish trunk. SUMMARYVasohibin-1 mediated detyrosination of endothelial microtubules is selectively required for adequate behaviour of venous secondary sprouting and subsequent formation of functional lymphatics in the zebrafish trunk.

developmental biology↗

bioRxiv2020

A Penicillium rubens platform strain for secondary metabolite production

We present a Penicillium rubens strain with an industrial background in which the four highly expressed biosynthetic gene clusters (BGC) required to produce penicillin, roquefortine, chrysogine and fungisporin were removed. This resulted in a minimal secondary metabolite background. Amino acid pools under steady-state growth conditions showed reduced levels of methionine and increased intracellular aromatic amino acids. Expression profiling of remaining BGC core genes and untargeted mass spectrometry did not identify products from uncharacterized BGCs. This platform strain was repurposed for expression of the recently identified polyketide calbistrin gene cluster and achieved high yields of decumbenone A, B and C. The penicillin BGC could be restored through in vivo assembly with eight DNA segments with short overlaps. Our study paves the way for fast combinatorial assembly and expression of biosynthetic pathways in a fungal strain with low endogenous secondary metabolite burden.

synthetic biology↗