Valorization of Cabbage Waste as a Feedstock for Microbial Polyhydroxyalkanoate Production: Optimizing Hydrolysis Conditions and Polyhydroxyalkanoate Production.

Establishing a platform for the bioconversion of waste resources into value-added compounds is critical for achieving a sustainable and eco-friendly economy. Herein, we produced polyhydroxyalkanoate via microbial fermentation using cabbage waste as a feedstock and metabolically engineered Escherichia coli. For this, the hydrolysis conditions of cabbage waste were optimized by focusing on parameters such as substrate and enzyme concentrations to enhance the saccharification efficiency. The phaABC operon, which encodes key enzymes responsible for polyhydroxyalkanoate biosynthesis in Ralstonia eutropha H16, was overexpressed in E. coli. Using cabbage hydrolysate as the feedstock, this engineered E. coli strain could produce poly(3-hydroxybutyrate) with a polymer content of 26.0 wt % of dry cell weight. Moreover, malic acid in cabbage hydrolysate significantly enhanced poly(3-hydroxybutyrate) production; the addition of 0.5 g/L malic acid markedly increased poly(3-hydroxybutyrate) content by 59.9%. This study demonstrates the potential of cabbage waste as a promising raw material for the microbial production of polyhydroxyalkanoate.

Heterotypic stress-induced adaptive evolution enhances freeze-drying tolerance and storage stability of Leuconostoc mesenteroides WiKim33.

Lactic acid bacteria (LAB) are currently being investigated for their potential use as probiotics and starter cultures. Researchers have developed powdering processes for the commercialization of LAB. Previous studies have focused on identifying innovative cryoprotective agents and freeze-drying (FD) techniques to enhance the stability of LAB. In this study, adaptive laboratory evolution (ALE) was employed to develop a strain with high FD tolerance and enhanced storage stability. Leuconostoc mesenteroids WiKim33 was subjected to heterotypic shock (heat and osmosis shock) to induce the desired phenotype and genotype. An FD-tolerant enhanced Leu. mesenteroides WiKim33 strain (ALE50) was obtained, which harbored a modified fatty acid composition and cell envelope characteristics. Specifically, ALE50 showed a lower unsaturated fatty acid (UFA)/saturated fatty acid (SFA) ratio and a higher cyclic fatty acid (CFA) composition. Moreover, the exopolysaccharide (EPS) thickness increased significantly by 331% compared to that of the wild type (WT). FD tolerance, which was evaluated using viability testing after FD, was enhanced by 33.4%. Overall, we demonstrated the feasibility of ALE to achieve desirable characteristics and provided insights into the mechanisms underlying increased FD tolerance.

Utilization of coffee waste as a sustainable feedstock for high-yield lactic acid production through microbial fermentation.

Lactic acid is an important industrial precursor; however, high substrate costs are a major challenge in microbial fermentation-based lactic acid production. Coffee waste is a sustainable feedstock alternative for lactic acid production via microbial fermentation. Herein, the feasibility of coffee waste as a feedstock was explored by employing appropriate pretreatment methods and optimizing enzyme combinations. Coffee waste pretreatment with hydrogen peroxide and acetic acid along with a combination of Viscozyme L, Celluclast 1.5 L, and Pectinex Ultra SP-L achieved the 78.9 % sugar conversion rate at a substrate concentration of 4 % (w/v). Lactiplantibacillus plantarum WiKim0126-induced fermentation with a 4 % solid loading yielded a lactic acid concentration of 22.8 g/L (99.6 % of the theoretical maximum yield) and productivity of 0.95 g/L/h within 24 h. These findings highlight the viability of coffee waste as an eco-friendly resource for sustainable lactic acid production.

Genome-wide identification of overexpression and downregulation gene targets based on the sum of covariances of the outgoing reaction fluxes.

In metabolic engineering, predicting gene overexpression targets remains challenging because both endogenous and heterologous genes in a large metabolic space can be candidates, in contrast to gene knockout targets that are confined to endogenous genes. We report the development of iBridge that identifies positive and negative metabolites exerting positive and negative impacts on product formation, respectively, based on the sum of covariances of their outgoing (consuming) reaction fluxes for a target chemical. Then, "bridge" reactions converting negative metabolites to positive metabolites are identified as overexpression targets, while the opposites as downregulation targets. Using iBridge, overexpression and downregulation targets are suggested for the production of 298 chemicals and validated for 36 chemicals experimentally demonstrated in previous studies. Finally, iBridge is employed to engineer Escherichia coli strains capable of producing 10.3 g/L of D-panthenol, a compound not previously produced, as well as putrescine and 4-hydroxyphenyllactate at enhanced titers, 63.7 and 8.3 g/L, respectively.

Sonochemical application reduces monosaccharide levels and improves cryoprotective effect of Jerusalem artichoke extract on Leuconostoc mesenteroides WiKim33 during freeze-drying.

Lactic acid bacteria (LAB) are being used for probiotic and starter cultures to prevent global damage to microbial cells. To retain the benefits of LAB in the commercially used powdered form, highly efficient cryoprotective agents are required during the manufacturing process. This study suggests a novel cryoprotective agent derived from Jerusalem artichoke (JA; Helianthus tuberous L.) and describes the mechanism of cryoprotective effect improvement by sonication treatment. The cryoprotective effect of JA extract was verified by examining the viability of Leuconostoc mesenteroides WiKim33 after freeze-drying (FD). Sonication of JA extract improved the cryoprotective effect. Sonication reduced fructose and glucose contents, which increased the induction of critical damage during FD by 15.84% and 46.81%, respectively. The cryoprotective effects of JA and sonication-treated JA extracts were determined using the viable cell count of Leu. mesenteroides WiKim33. Immediately after FD and storage for 24 weeks, the viability of Leu. mesenteroides WiKim33 with JA extract was 82.8% and 76.3%, respectively, while that of the sonication-treated JA extract was 95.2% and 88.8%, respectively. Our results show that reduction in specific monosaccharides was correlated with improved cryoprotective effect. This study adopted sonication as a novel treatment for improving the cryoprotective effect and verified its efficiency.

Use of Vegetable Waste as a Culture Medium Ingredient Improves the Antimicrobial and Immunomodulatory Activities of Lactiplantibacillus plantarum WiKim0125 Isolated from Kimchi.

Lactic acid bacteria (LAB) isolated from kimchi (a traditional Korean dish typically made of fermented cabbage) can provide various health benefits, including anti-obesity, antioxidant, anti-inflammatory, anticancer, and antimicrobial effects. In this study, we examined the antimicrobial and immunomodulatory effects of Lactiplantibacillus plantarum WiKim0125 cultured in de Man, Rogosa, and Sharpe (MRS) medium containing vegetable waste. Live bacterial cells were eliminated via supernatant filtration or heat treatment. The cell-free supernatant (CFS) obtained from culture broth containing kimchi cabbage waste (KCW), cabbage waste (CW), or onion waste (OW) showed significantly higher antimicrobial activity against skin pathogens (Propionibacterium acnes and Staphylococcus aureus) and foodborne pathogens (Escherichia coli and Salmonella typhimurium), with inhibition zones ranging between 4.4 and 8.5 mm, compared to that in conventional MRS medium (4.0-7.3 mm). In lipopolysaccharide-stimulated RAW264.7 cells, both supernatant and heat-inactivated Lb. plantarum WiKim0125 from culture media containing KCW and CW suppressed the production of inflammatory cytokines (72.8% and 49.6%, respectively) and nitric oxide (62.2% and 66.7%, respectively) without affecting cell viability. These results indicate that vegetable waste can potentially increase the antimicrobial and immunoregulatory potency of LAB while presenting a molecular basis for applying postbiotics to health products.

Rapid and Efficient Removal of Anionic Dye in Water Using a Chitosan-Coated Iron Oxide-Immobilized Polyvinylidene Fluoride Membrane.

Anionic dyes are one of the most serious contaminants in water as these molecules are known to be toxic to many living organisms. Herein, we report the development of functionalized polyvinylidene fluoride membranes modified with chitosan-coated iron oxide nanomaterials (Fe-PVDF) for the efficient treatment of anionic dye-contaminated water. Aqueous solutions of anionic dyes could be captured rapidly by passing through the functionalized membrane under reduced pressure. Under neutral conditions, Fe-PVDF showed a maximum removal capacity of 74.6 mg/g for Evans blue (EB) through the adsorption process. In addition, the adsorption capacity was significantly enhanced up to 434.78 mg/g under acidic conditions. The adsorption process for EB matched well with the Langmuir model, indicating monolayer adsorption of the dye to the membrane surface. Moreover, Fe-PVDF can be reusable by a simple washing step in an alkaline solution, and thus, the composite membrane was applied several times without a significant decrease in its adsorption performance. The same composite membrane was further applied to the removal of five other different anionic dyes with high efficiencies. The adsorption mechanism can be explained by the electrostatic interaction between the positively charged chitosan and the negatively charged dye as well as the affinity of the sulfate groups in dye molecules for the surface of the iron oxide nanoparticles. The easy preparation and rapid decolorization procedures make this composite membrane suitable for efficient water treatment.

Isolation and Characterization of a Yellow Xanthophyll Pigment-Producing Marine Bacterium, Erythrobacter sp. SDW2 Strain, in Coastal Seawater.

Xanthophylls, a yellow pigment belonging to the carotenoid family, have attracted much attention for industrial applications due to their versatile nature. We report the isolation of a homo xanthophyll pigment-producing marine bacterium, identified as the Erythrobacter sp. SDW2 strain, from coastal seawater. The isolated Erythrobacter sp. SDW2 strain can produce 263 ± 12.9 mg/L (89.7 ± 5.4 mg/g dry cell weight) of yellow xanthophyll pigment from 5 g/L of glucose. Moreover, the xanthophyll pigment produced by the SDW2 strain exhibits remarkable antioxidative activities, confirmed by the DPPH (73.4 ± 1.4%) and ABTS (84.9 ± 0.7%) assays. These results suggest that the yellow xanthophyll pigment-producing Erythrobacter sp. SDW2 strain could be a promising industrial microorganism for producing marine-derived bioactive compounds with potential for foods, cosmetics, and pharmaceuticals.

Structural and functional characterization of a monoclonal antibody blocking TIGIT.

TIGIT is an immune checkpoint receptor that is expressed on subsets of activated T cells and natural killer (NK) cells. Several ligands for TIGIT, including poliovirus receptor (PVR), are expressed on cancer cells and mediate inhibitory signaling to suppress antitumor activities of the immune cells. Many studies support that the TIGIT signaling is a potential target for cancer immunotherapy. We developed an IgG4-type monoclonal antibody against human TIGIT, designated as MG1131, using a phage display library of single-chain variable fragments (scFvs). MG1131 interacts with TIGIT much more tightly than PVR does. The crystal structure of a scFv version of MG1131 bound to TIGIT was determined, showing that MG1131 could block the PVR-TIGIT interaction and thus the immunosuppressive signaling of TIGIT. Consistently, MG1131 is bound to TIGIT-expressing cells and interferes with PVR binding to these cells. Moreover, MG1131 increased NK cell-mediated tumor killing activities, inhibited immunosuppressive activity of regulatory T (Treg) cells from healthy donors, and restored interferon-γ secretion from peripheral blood mononuclear cells derived from multiple myeloma patients. MG1131 also increased T cell infiltration to the tumor site and inhibited tumor growth in mice. Collectively, these data indicate that MG1131 modulates the effector functions of T cells and NK cells positively and Treg cells negatively.

The Protective Effect of Edible Bird's Nest against the Immune-senescence Process of UVB-irradiated Hairless Mice.

Edible bird's nest (EBN) is a nutritious food with many beneficial effects, including protecting cells against oxidation and infection due to wounds, bacteria or viruses. EBN has shown antiaging, anti-inflammatory and wound-healing properties in skin cells. Here, we investigated whether EBN has protective effects against photoaging, inflammation and immune-senescence in hairless mice treated with UVB irradiation. The skin thickness was lower in mice on an EBN diet than in mice treated with UVB alone. The level of hydration was significantly increased, while the index of transepidermal water loss decreased, in groups on the EBN diet. EBN also reduced erythema index in UVB-irradiated mice. At the molecular level, skin elasticity and antiaging are associated with high expression of elastin, collagen and filaggrin and low expression of the membrane metalloproteinases, MMP-1 and MMP-2. Inflammatory markers such as interleukins, IL-1ß and IL-6, and TNF-α decreased significantly in the EBN groups. Caspase-3, an important factor in the apoptotic pathway and in congenital and adaptive immune responses, decreased in the EBN groups. Moreover, EBN diminished the overexpression of immunoglobulin E and elevated mast cell counts in UVB-irradiated mice. Overall, these findings suggest that EBN protects skin against aging and alleviates inflammation in UVB-irradiated hairless mice.

Dysfunction of NMDA receptors in neuronal models of an autism spectrum disorder patient with a DSCAM mutation and in Dscam-knockout mice.

Heterogeneity in the etiopathology of autism spectrum disorders (ASD) limits the development of generic remedies, requires individualistic and patient-specific research. Recent progress in human-induced pluripotent stem cell (iPSC) technology provides a novel platform for modeling ASDs for studying complex neuronal phenotypes. In this study, we generated telencephalic induced neuronal (iN) cells from iPSCs derived from an ASD patient with a heterozygous point mutation in the DSCAM gene. The mRNA of DSCAM and the density of DSCAM in dendrites were significantly decreased in ASD compared to control iN cells. RNA sequencing analysis revealed that several synaptic function-related genes including NMDA receptor subunits were downregulated in ASD iN cells. Moreover, NMDA receptor (R)-mediated currents were significantly reduced in ASD compared to control iN cells. Normal NMDA-R-mediated current levels were rescued by expressing wild-type DSCAM in ASD iN cells, and reduced currents were observed by truncated DSCAM expression in control iN cells. shRNA-mediated DSCAM knockdown in control iN cells resulted in the downregulation of an NMDA-R subunit, which was rescued by the overexpression of shRNA-resistant DSCAM. Furthermore, DSCAM was co-localized with NMDA-R components in the dendritic spines of iN cells whereas their co-localizations were significantly reduced in ASD iN cells. Levels of phospho-ERK1/2 were significantly lower in ASD iN cells, suggesting a potential mechanism. A neural stem cell-specific Dscam heterozygous knockout mouse model, showing deficits in social interaction and social memory with reduced NMDA-R currents. These data suggest that DSCAM mutation causes pathological symptoms of ASD by dysregulating NMDA-R function.

Production, Characterization, and Antioxidant Activities of an Exopolysaccharide Extracted from Spent Media Wastewater after Leuconostoc mesenteroides WiKim32 Fermentation.

Bacterial exopolysaccharides (EPSs) are important alternatives to plant polysaccharides in fermented products and exhibit antioxidant activity, which is particularly desirable for functional foods. This study evaluated the use of spent media wastewater (SMW) derived from kimchi fermentation for the production of an EPS and analyzed the characterization and antioxidant activity of the resulting EPS. The EPS concentration and conversion yields of sequential purification were 7.7-9.0 g/L and 38.6-45.1%, respectively. Fourier transform infrared spectra and NMR spectra indicated that the EPS was a linear glucan with α-(1 → 6) linkages. The EPS also exhibited thermal tolerance to high temperatures. In vitro antioxidant activity analyses indicated the scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, thiobarbituric acid reactance (TBAR), and ferric ion reducing antioxidant power (FRAP) values of 71.6-79.1, 28.2-33.0%, and 0.04-0.05 mM FeCl3, respectively. These results reveal that the EPS extracted from SMW has potential as a thermally tolerant, nontoxic, and natural antioxidant for industrial applications.

Variable Tumor Microenvironment-on-a-chip with Temporal Angiogenic Switching System by Diffusion Control.

Organ-on-a-chip has the potential to replace preclinical trials which have been problematic for decades due to unaffordable cost and time. The performance of in vitro tumor-on-a-chip depends on how accurately the system represents analogous tumor-microenvironment (TME) and TME associated phenomena. In this study, we have focused on angiogenesis, one of the most significant features of TME for tumor growth and metastasis. Angiogenesis in TME is triggered through cascaded interactions among TME associated neighboring cells including immune cells, tumor cells, and fibroblast cells [1]. Therefore, temporally-controlled TME-on-a-chip is desired for an accurate representation of angiogenesis. However, conventional microfluidic devices cannot temporarily manipulate the condition of interacting cells and secreted signal molecules. Here, we proposed a hydrogel-based variable TME-on-a-chip with diffusion switch channels. The channels between hydrogel walls enable temporal diffusion control by controlling inflow. The diffusion control was observed in diffusion experiment with a fluorescent dye. Furthermore, experiment of HUVEC's migration toward diffused VEGF also confirmed that TME-on-a-chip is capable of reproducing an angiogenic switch triggering through temporal diffusion control. Due to a simple fabrication procedure, the design of the microfluidic device can be easily modified to represent more complex variable TME models.

Mechanisms of Insecticidal Action of Metarhizium anisopliae on Adult Japanese Pine Sawyer Beetles (Monochamus alternatus).

Pine wilt disease, caused by Bursaphelenchus xylophilus (pine wood nematode), leads to severe environmental and economic damage. Here, we report the results of experiments on the biological control of pine wilt disease through termination of the insect vector of the nematode and the mechanism of the insecticidal action of Metarhizium anisopliae JEF-279 against Monochamus alternatus (Japanese pine sawyer). A combined treatment with a fungal conidia suspension and a fungal protease-containing culture filtrate caused 75.8% mortality of the insect vector. Additionally, the presence of destruxins was confirmed in the dead Japanese pine sawyer adults, and half of the 10 protein spots in proteomic analysis were identified as an actin related to muscle contraction. Based on proteomic and microscopic analyses, the infection cycle of the Japanese pine sawyer by M. anisopliae JEF-279 was inferred to proceed in the following sequence: (1) host adhesion and germination, (2) epicuticle degradation, (3) growth as blastospore, (4) killing by various fungal toxins (insecticidal metabolites), (5) immune response as defense mechanism, and (6) hyphal extrusion and conidiation. Consequently, the combined fungal conidia suspension and protease-containing culture filtrate treatment may be applied as an insecticidal agent, and flaccid paralysis is likely a major mechanism underlying the insecticidal action of M. anisopliae JEF-279 on host insects.

Enhanced Lycopene Production by UV-C Irradiation in Radiation-Resistant Deinococcus radiodurans R1.

Although classical metabolic engineering strategies have succeeded in developing microbial strains capable of producing desired bioproducts, metabolic imbalance resulting from extensive genetic manipulation often leads to decreased productivity. Thus, abiotic strategies for improving microbial production performance can be an alternative to overcome drawbacks arising from intensive metabolic engineering. Herein, we report a promising abiotic method for enhancing lycopene production by UV-C irradiation using a radiation-resistant ΔcrtLm/crtB+dxs+ Deinococcus radiodurans R1 strain. First, the onset of UV irradiation was determined through analysis of the expression of 11 genes mainly involved in the carotenoid biosynthetic pathway in the ΔcrtLm/crtB+dxs+ D. radiodurans R1 strain. Second, the effects of different UV wavelengths (UV-A, UV-B, and UV-C) on lycopene production were investigated. UV-C irradiation induced the highest production, resulting in a 69.9% increase in lycopene content [64.2 ± 3.2 mg/g dry cell weight (DCW)]. Extended UV-C irradiation further enhanced lycopene content up to 73.9 ± 2.3 mg/g DCW, a 95.5% increase compared to production without UV-C irradiation (37.8 ± 0.7 mg/g DCW).

Coffee residue as a valorization bio-agent for shelf-life extension of lactic acid bacteria under cryopreservation.

The present work describes the feasibility of coffee residue extracts as cryoprotective agents in the storage stability of freeze-dried lactic acid bacteria. Coffee residue extracts were extracted from coffee residue, produced after coffee extraction for coffee powder and instant coffee preparation, using an autoclave. Leuconostoc mesenteroides WiKim32 was selected to evaluate the ability of coffee residue extracts to protect bacteria during freeze-dried storage. The storage stability of freeze-dried Leu. mesenteroides WiKim32 with coffee residue extracts was comparable to those with commercial cryoprotective agents. Coffee residue extracts contributed to storage stability immediately after freeze-drying (61.2%) and subsequent storage (48.7%). Our data indicate that the protective effect of the coffee residue extracts is associated with ions, carbohydrates, and phenolic compounds. Coffee residue extracts are feasible materials, which can reduce the storage and distribution costs compared to commercial agents currently available.

Mixture of enzyme-processed Panax ginseng and Gastrodia elata extract prevents UVB-induced decrease of procollagen type 1 and increase of MMP-1 and IL-6 in human dermal fibroblasts.

According to the previously described anti-photoaging effect of the enzyme-processed Panax ginseng extract and Gastrodia elata extract, we hypothesized that the combination of the two extracts would have superior effect to protect human skin from UVB radiation. Besides, the mixture of active components isolated from herbal extracts, ginsenoside F2, and α-gastrodin was investigated on the photo-protective capability. The expression of aging-related markers including matrix metalloproteinase-1 (MMP-1), interleukin-6 (IL-6), and procollagen type 1 was evaluated using ELISA kits. It was reported that the herbal extract at a Panax ginseng extract to Gastrodia elata extract ratio of 1:10 (w/w) and the compound mixture with equal proportion of ginsenoside F2 and α-gastrodin exhibited significant inhibition of MMP-1 and IL-6 production, and marked upregulation of procollagen type 1 formation. Thus, the combination of either the enzyme-processed herbal extracts or their active components would enhance the properties of prevention and treatment of UVB-induced skin damage.

Cohen Syndrome Patient iPSC-Derived Neurospheres and Forebrain-Like Glutamatergic Neurons Reveal Reduced Proliferation of Neural Progenitor Cells and Altered Expression of Synapse Genes.

Cohen syndrome (CS), a rare autosomal recessive disorder, has been associated with genetic mutations in the VPS13B gene, which regulates vesicle-mediated protein sorting and transport. However, the cellular mechanism underlying CS pathogenesis in patient-derived human neurons remains unknown. We identified a novel compound heterozygous mutation, due to homozygous variation of biparental origin and heterozygous variation inherited from the father, in the VPS13B gene in a 20-month-old female patient. To understand the cellular pathogenic mechanisms, we generated induced pluripotent stem cells (iPSCs) from the fibroblasts of the CS patient. The iPSCs were differentiated into forebrain-like functional glutamatergic neurons or neurospheres. Functional annotation from transcriptomic analysis using CS iPSC-derived neurons revealed that synapse-related functions were enriched among the upregulated and downregulated genes in the CS neurons, whereas processes associated with neurodevelopment were enriched in the downregulated genes. The developing CS neurospheres were small in size compared to control neurospheres, likely due to the reduced proliferation of SOX2-positive neural stem cells. Moreover, the number of SV2B-positive puncta and spine-like structures was significantly reduced in the CS neurons, suggesting synaptic dysfunction. Taking these findings together, for the first time, we report a potential cellular pathogenic mechanism which reveals the alteration of neurodevelopment-related genes and the dysregulation of synaptic function in the human induced neurons differentiated from iPSCs and neurospheres of a CS patient.

High-Yield Production of Lycopene from Corn Steep Liquor and Glycerol Using the Metabolically Engineered Deinococcus radiodurans R1 Strain.

Developing a highly efficient and ecofriendly system to produce desired products from waste can be considered important to a sustainable society. Here, we report for the first time high-yield production of lycopene through metabolically engineering an extremophilic microorganism, Deinococcus radiodurans R1, from corn steep liquor (CSL) and glycerol. First, the crtLm gene-encoding lycopene cyclase was deleted to prevent the conversion of lycopene to γ-carotene. Then, the crtB gene-encoding phytoene synthase and the dxs gene-encoding 1-deoxy-d-xylulose 5-phosphate synthase were overexpressed to increase carbon flux toward lycopene. The engineered ΔcrtLm/crtB+dxs+ D. radiodurans R1 could produce 273.8 mg/L [80.7 mg/g dry cell weight (DCW)] and 373.5 mg/L (108.0 mg/g DCW) of lycopene from 10 g/L of glucose with 5 g/L of yeast extract and 9.9 g/L of glucose with 20 g/L of CSL, respectively. Moreover, the lycopene titer and content were increased by 26% (470.6 mg/L) and 28% (138.2 mg/g DCW), respectively, when the carbon source was changed to glycerol. Finally, fed-batch fermentation of the final engineered strain allowed the production of 722.2 mg/L (203.5 mg/g DCW) of lycopene with a yield and productivity of 20.3 mg/g glycerol and 6.0 mg/L/h, respectively, from 25 g/L of CSL and 35.7 g/L of glycerol.

Identification of a novel Shank2 transcriptional variant in Shank2 knockout mouse model of autism spectrum disorder.

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders that are highly heterogeneous in clinical symptoms as well as etiologies. Mutations in SHANK2 are associated with ASD and accordingly, Shank2 knockout mouse shows ASD-like behavioral phenotypes, including social deficits. Intriguingly, two lines of Shank2 knockout (KO) mouse generated by deleting different exons (exon 6-7 or exon 7) showed distinct cellular phenotypes. Previously, we compared gene expressions between Shank2 KOs lacking exon 6-7 (e6-7 KO) and KOs lacking exon 7 (e7 KO) by performing RNA-seq. In this study, we expanded transcriptomic analyses to identify novel transcriptional variants in the KO mice. We found prominent expression of a novel exon (exon 4' or e4') between the existing exons 4 and 5 in the Shank2 e6-7 KO model. Expression of the transcriptional variant harboring this novel exon was confirmed by RT-PCR and western blotting. These findings suggest that the novel variant may function as a modifier gene, which contributes to the differences between the two Shank2 mutant lines. Furthermore, our result further represents an example of genetic compensation that may lead to phenotypic heterogeneity among ASD patients with mutations in the same gene.