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Sustainable production of edible microbial proteins and red food colorants is an important demand for future food. Therefore, creation of a chassis strain that can efficiently synthesize both products is extremely necessary and meaningful. To realize this envision, a CRISPR/Cas9-based visual multicopy integration system was successfully developed in Fusarium venenatum. Subsequently, the de novo synthesis of the red food colorant betanin was achieved in the engineered F. venenatum using the above system. After fermentation optimization, the final yields of betanin and mycoprotein reached 1.91 and 9.53 g/L, respectively, when the constant pH naturally decreased from 6 to 4 without the addition of acid after 48 h of fermentation. These results determine a highly suitable chassis strain for the microbial biomanufacturing of betanin, and the obtained engineered strain here is expected to expand the application prospect and improve economic returns of F. venenatum in the field of future food.

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OBJECTIVES: This study investigated the codigestion of corn straw (CS) with cow manure (CM), cow digestion solution (CD), and a strain consortium (SC) for enhanced volatile fatty acid (VFA) production. The aims of this study were to develop a sustainable technique to increase VFA yields, examine how combining microbial reagents with CS affects VFA production by functional microorganisms, and assess the feasibility of improving microbial diversity through codigestion. METHODS: Batch experiments evaluated VFA production dynamics and microbial community changes with different combinations of CS substrates with CM, CD, and SC. Analytical methods included measuring VFAs by GC, ammonia and chemical oxygen demand (COD) by standard methods and microbial community analysis by 16S rRNA gene sequencing. RESULTS: Codigesting CS with the strain consortium yielded initial VFA concentrations ranging from 0.6 to 1.0 g/L, which were greater than those of the other combinations (0.05-0.3 g/L). Including CM, and CD further increased VFA production to 1.0-2.0 g/L, with the highest value of 2.0 g/L occurring when all four substrates were codigested. Significant ammonium reduction (194-241 mg/L to 29-37 mg/L) and COD reduction (3310-5250 mg/L to 730-1210 mg/L) were observed. Codigestion with CM and CD had greater Shannon diversity indices (3.19-3.24) than did codigestion with the other consortia (2.26). Bacillota dominated (96.5-99.6 %), with Clostridiales playing key roles in organic matter breakdown. CONCLUSIONS: This study demonstrated the feasibility of improving VFA yields and harnessing microbial diversity through anaerobic codigestion of lignocellulosic and animal waste streams. Codigestion substantially enhanced VFA production, which was dominated by butyrate, reduced ammonium and COD, and enriched fiber-degrading and fermentative bacteria. These findings can help optimize codigestion for sustainable waste management and high-value chemical production.

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After the second industrial revolution, social productivity developed rapidly, and the use of fossil fuels such as coal, oil, and natural gas increased greatly in industrial production. The burning of these fossil fuels releases large amounts of greenhouse gases such as CO2, which has caused greenhouse effects and global warming. This has endangered the planet's ecological balance and brought many species, including animals and plants, to the brink of extinction. Thus, it is crucial to address this problem urgently. One potential solution is the use of syngas fermentation with microbial cell factories. This process can produce chemicals beneficial to humans, such as ethanol as a fuel while consuming large quantities of harmful gases, CO and CO2. However, syngas-fermenting microorganisms often face a metabolic energy deficit, resulting in slow cell growth, metabolic disorders, and low product yields. This problem limits the large-scale industrial application of engineered microorganisms. Therefore, it is imperative to address the energy barriers of these microorganisms. This paper provides an overview of the current research progress in addressing energy barriers in bacteria, including the efficient capture of external energy and the regulation of internal energy metabolic flow. Capturing external energy involves summarizing studies on overexpressing natural photosystems and constructing semiartificial photosynthesis systems using photocatalysts. The regulation of internal energy metabolic flows involves two parts: regulating enzymes and metabolic pathways. Finally, the article discusses current challenges and future perspectives, with a focus on achieving both sustainability and profitability in an economical and energy-efficient manner. These advancements can provide a necessary force for the large-scale industrial application of syngas fermentation microbial cell factories.

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Nitrogen and carbon are the two most essential nutrient elements, and their metabolism is tightly coupled in single carbon metabolic microorganisms. However, the nitrogen metabolism and the nitrogen/carbon (N/C) metabolic balance in single-carbon metabolism is poorly studied. In this study, the nitrogen metabolism pattern of the fast growing methanotrophs Methylomonas sp. ZR1 grown in methane and methanol was studied. Effect study of different nitrogen sources on the cell growth of ZR1 indicates that nitrate salts are the best nitrogen source supporting the growth of ZR1 using methane and methanol as carbon source. However, its metabolic intermediate ammonium was found to accumulate with high N/C ratio in the medium and consequently inhibit the growth of ZR1. Studies of carbon and nitrogen metabolic kinetic under different N/C ratio conditions indicate that the accumulation of NH4+ is caused by the imbalanced nitrogen and carbon metabolism in ZR1. Feeding carbon skeleton α-ketoglutaric acid could effectively relieve the inhibition effect of NH4+ on the growth of ZR1, which further confirms this assumption. qPCR analysis of the expression level of the central metabolic key enzyme gene indicates that the nitrogen metabolic intermediate ammonium has strong regulation effect on the central nitrogen and carbon metabolism in ZR1. qPCR-combined genomic analysis confirms that a third ammonium assimilation pathway glycine synthesis system is operated in ZR1 to balance the nitrogen and carbon metabolism. Based on the qPCR result, it was also found that ZR1 employs two strategies to relieve ammonium stress in the presence of ammonium: assimilating excess ammonium or cutting off the nitrogen reduction reactions according to the available C1 substrate. Validating the connections between single-carbon and nitrogen metabolism and studying the accumulation and assimilation mechanism of ammonium will contribute to understand how nitrogen regulates cellular growth in single-carbon metabolic microorganisms.(AU)

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Nitrogen and carbon are the two most essential nutrient elements, and their metabolism is tightly coupled in single carbon metabolic microorganisms. However, the nitrogen metabolism and the nitrogen/carbon (N/C) metabolic balance in single-carbon metabolism is poorly studied. In this study, the nitrogen metabolism pattern of the fast growing methanotrophs Methylomonas sp. ZR1 grown in methane and methanol was studied. Effect study of different nitrogen sources on the cell growth of ZR1 indicates that nitrate salts are the best nitrogen source supporting the growth of ZR1 using methane and methanol as carbon source. However, its metabolic intermediate ammonium was found to accumulate with high N/C ratio in the medium and consequently inhibit the growth of ZR1. Studies of carbon and nitrogen metabolic kinetic under different N/C ratio conditions indicate that the accumulation of NH4+ is caused by the imbalanced nitrogen and carbon metabolism in ZR1. Feeding carbon skeleton α-ketoglutaric acid could effectively relieve the inhibition effect of NH4+ on the growth of ZR1, which further confirms this assumption. qPCR analysis of the expression level of the central metabolic key enzyme gene indicates that the nitrogen metabolic intermediate ammonium has strong regulation effect on the central nitrogen and carbon metabolism in ZR1. qPCR-combined genomic analysis confirms that a third ammonium assimilation pathway glycine synthesis system is operated in ZR1 to balance the nitrogen and carbon metabolism. Based on the qPCR result, it was also found that ZR1 employs two strategies to relieve ammonium stress in the presence of ammonium: assimilating excess ammonium or cutting off the nitrogen reduction reactions according to the available C1 substrate. Validating the connections between single-carbon and nitrogen metabolism and studying the accumulation and assimilation mechanism of ammonium will contribute to understand how nitrogen regulates cellular growth in single-carbon metabolic microorganisms.

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The global protein shortage is intensifying, and promising means to ensure daily protein supply are desperately needed. The mycoprotein produced by Fusarium venenatum is a good alternative to animal/plant-derived protein. To comprehensively improve the mycoprotein synthesis, a stepwise strategy by blocking the byproduct ethanol synthesis and the gluconeogenesis pathway and by optimizing the fermentation medium was herein employed. Ultimately, compared to the wild-type strain, the synthesis rate, carbon conversion ratio, and protein content of mycoprotein produced from the engineered strain were increased by 57% (0.212 vs 0.135 g/L·h), 62% (0.351 vs 0.217 g/g), and 57% (61.9 vs 39.4%), respectively, accompanied by significant reductions in CO2 emissions. These results provide a referential strategy that could be useful for improving mycoprotein synthesis in other fungi; more importantly, the obtained high-mycoprotein-producing strain has the potential to promote the development of the edible protein industry and compensate for the gap in protein resources.

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Aronia melanocarpa (Michx.) Ell. is a rich source of anthocyanins and proanthocyanidins with confirmed health benefits. Individual cyanidin glucosides (cyanidin 3-galactoside, cyanidin 3-arabinoside, cyanidin 3-xyloside, and cyanidin 3-glucoside) of anthocyanins (calculated by individual cyanin glycoside fractions was 419.9 mg/100 g FW) were isolated by Sephadex LH-20 column and different parts of proanthocyanidins with a different mean degree of polymerization (mDP) were fractionated by the solubility differences in different solvents. The composition of different mDP of proanthocyanidins was as follows: monomers (1.51%), oligomer (mDP of 4.2 ± 0.9, 20.57%), CPP-50 (mDP of 78.9 ± 4.1, 22.17%), CPP-60 (mDP of 66.1 ± 1.2, 27.94%), CPP-70 (mDP of 36.8 ± 3.9, 36.8%), CPP-75 (mDP of 25.2 ± 1.3, 6.14%), CPP-L (mDP of 10.2 ± 2.6, 6.95%), and there were recycling loss of 0.34%. Cyanidin 3-glucoside showed the strongest inhibition effects on α-amylase and lipase and cyanidin 3-arabinoside showed the strongest inhibition effect on α-glucosidase, while cyanidin 3-xyloside has no inhibition effect on the α-amylase, and cyanidin 3-galactoside, cyanidin 3-arabinoside, and cyanidin 3-xyloside have no inhibition effects on lipase. The inhibition effect of proanthocyanidins with different mDP to the enzymes all showed high negative correlations between the mDP and IC50 (half-maximal inhibitory concentration). This study suggests that A. melanocarpa (Michx.) Ell. can have beneficial effects due to inhibition of the digestion enzyme.

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Esophageal cancer has become a malignant tumor disease with high mortality worldwide. Many cases of esophageal cancer are not very serious in the beginning but become severe in the late stage, so the best treatment time is missed. Less than 20% of patients with esophageal cancer are in the late stage of the disease for 5 years. The main treatment method is surgery, which is assisted by radiotherapy and chemotherapy. Radical resection is the most effective treatment method, but a method for imaging examination of esophageal cancer with good clinical effect has yet to be developed. This study compared imaging staging of esophageal cancer with pathological staging after operation based on the big data of intelligent medical treatment. MRI can be used to evaluate the depth of esophageal cancer invasion and replace CT and EUS for accurate diagnosis of esophageal cancer. Intelligent medical big data, medical document preprocessing, MRI imaging principal component analysis and comparison and esophageal cancer pathological staging experiments were used. Kappa consistency tests were conducted to compare the consistency between MRI staging and pathological staging and between two observers. Sensitivity, specificity and accuracy were determined to evaluate the diagnostic effectiveness of 3.0T MRI accurate staging. Results showed that 3.0T MR high-resolution imaging could show the histological stratification of the normal esophageal wall. The sensitivity, specificity and accuracy of high-resolution imaging in staging and diagnosis of isolated esophageal cancer specimens reached 80%. At present, preoperative imaging methods for esophageal cancer have obvious limitations, while CT and EUS have certain limitations. Therefore, non-invasive preoperative imaging examination of esophageal cancer should be further explored.Esophageal cancer has become a malignant tumor disease with high mortality worldwide. Many cases of esophageal cancer are not very serious in the beginning but become severe in the late stage, so the best treatment time is missed. Less than 20% of patients with esophageal cancer are in the late stage of the disease for 5 years. The main treatment method is surgery, which is assisted by radiotherapy and chemotherapy. Radical resection is the most effective treatment method, but a method for imaging examination of esophageal cancer with good clinical effect has yet to be developed. This study compared imaging staging of esophageal cancer with pathological staging after operation based on the big data of intelligent medical treatment. MRI can be used to evaluate the depth of esophageal cancer invasion and replace CT and EUS for accurate diagnosis of esophageal cancer. Intelligent medical big data, medical document preprocessing, MRI imaging principal component analysis and comparison and esophageal cancer pathological staging experiments were used. Kappa consistency tests were conducted to compare the consistency between MRI staging and pathological staging and between two observers. Sensitivity, specificity and accuracy were determined to evaluate the diagnostic effectiveness of 3.0T MRI accurate staging. Results showed that 3.0T MR high-resolution imaging could show the histological stratification of the normal esophageal wall. The sensitivity, specificity and accuracy of high-resolution imaging in staging and diagnosis of isolated esophageal cancer specimens reached 80%. At present, preoperative imaging methods for esophageal cancer have obvious limitations, while CT and EUS have certain limitations. Therefore, non-invasive preoperative imaging examination of esophageal cancer should be further explored.

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CRISPR/Cas9-mediated homology-directed recombination is an efficient method to express target genes. Based on the above method, providing ideal neutral integration sites can ensure the reliable, stable, and high expression of target genes. In this study, we obtained a fluorescent transformant with neutral integration and high expression of the GFP expression cassette from the constructed GFP expression library and named strain FS. The integration site mapped at 4886 bp upstream of the gene FVRRES_00686 was identified in strain FS based on a Y-shaped adaptor-dependent extension, and the sequence containing 600 bp upstream and downstream of this site was selected as the candidate region for designing sgRNAs (Sites) for CRISPR/Cas9-mediated homology-directed recombination. PCR analysis showed that the integration efficiency of CRISPR/Cas9-mediated integration of target genes in designed sites reached 100%. Further expression stability and applicability analysis revealed that the integration of the target gene into the above designed sites can be stably inherited and expressed and has no negative effect on the growth of F. venenatum TB01. These results indicate the above designed neutral sites have the potential to accelerate the development of F. venenatum TB01 through overexpression of target genes in metabolic engineering.

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Food scarcity and environmental deterioration are two major problems that human populations currently face. Fortunately, the disruptive innovation of raw food materials has been stimulated by the rapid evolution of biomanufacturing. Therefore, it is expected that the new trends in technology will not only alter the natural resource-dependent food production systems and the traditional way of life but also reduce and assimilate the greenhouse gases released into the atmosphere. This review article summarizes the metabolic pathways associated with C1 gas conversion and the production of single-cell protein for animal feed. Moreover, the protein function, worldwide authorization, market access, and methods to overcome challenges in C1 gas assimilation microbial cell factory construction are also provided. With widespread attention and increasing policy support, the production of C1 gas protein will bring more opportunities and make tremendous contributions to our sustainable future.

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Astaxanthin (3,3'-dihydroxy-4,4'-diketo-ß-carotene) is a type of C40 carotenoid with remarkable antioxidant characteristics, showing significant application prospects in many fields. Traditionally, the astaxanthin is mainly obtained from chemical synthesis and natural acquisition, with both approaches having many limitations and not capable of meeting the growing market demand. In order to cope with these challenges, novel techniques, e.g., the innovative cell engineering strategies, have been developed to increase the astaxanthin production. In this review, we first elaborated the biosynthetic pathway of astaxanthin, with the key enzymes and their functions discussed in the metabolic process. Then, we summarized the conventional, non-genetic strategies to promote the production of astaxanthin, including the methods of exogenous additives, mutagenesis, and adaptive evolution. Lastly, we reviewed comprehensively the latest studies on the synthesis of astaxanthin in various recombinant microorganisms based on the concept of microbial cell factory. Furthermore, we have proposed several novel technologies for improving the astaxanthin accumulation in several model species of microorganisms.

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Lactic acid bacteria (LAB) have exhibited strain/species specificity for different food matrices. We investigated the impact of LAB fermentation on the flavor, chemical profile, and bioactivity of goji juice. The colony counts of five selected strains reached above 8.5 log CFU/mL. The fermentation increased the organic acids, decreased the sugars, and improved the sensory quality of goji juice. The majority of the strains had increased acetic acid, heptanoic acid, ethyl phenylacetate, and linalool levels. Specific strains suppressed α-glucosidase and pancreatic lipase activities and increased the antioxidant activities of fermented goji juice. Based on non-targeted metabolomics and activities, 23 important differential metabolites were screened among 453 metabolites. The quantification results showed that isoquercitrin and m-coumaric content varied among strains, reflecting the strain specificity in flavone and flavonol biosynthesis and phenylalanine, tyrosine, and tryptophan biosynthesis. These findings will provide useful information for fermented goji juice biochemistry research.

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BACKGROUND: Rice straw (RS) is one of the largest sources of lignocellulosic, which is an abundant raw material for biofuels and chemicals. However, the natural degradation of RS under a low temperature environment is the biggest obstacle to returning straw to the field. RESULTS: In the present study, one bacillus strain W118 was isolated. Strain W118 was identified as Bacillus cereus through morphological and physiological characterization and 16S rDNA sequencing. The optimum growth temperature and pH of strain W118 were 20 °C and 6.5, respectively. Simultaneously, it was found that the strain W118 grew well at low temperature, even at a temperature of 4 °C (OD600  = 1.40 ± 0.01). The decrease of various compositions of RS after the fermentation process at a temperature of 20 °C and 4 °C for 14 days was 27.00 ± 0.02% and 23.70 ± 0.04%, respectively. The composition of RS decreased to 50.71 ± 0.02% after being fermented at 4 °C for 25 days. The results of scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction of RS showed that the compositions of RS were significant decreased. CONCLUSION: This test suggests that the strain W118 is efficient for degrading RS at low temperature, which has great application potential for straw degradation in a low temperature area. © 2022 Society of Chemical Industry.

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In order to alleviate the pressure on environmental resources faced by meat and dairy production and to satisfy the increasing demands of consumers for food safety and health, alternative proteins have drawn considerable attention in the food industry. However, despite the successive reports of alternative protein food, the processing and application foundation of alternative proteins for meat and dairy is still weak. This paper summarizes the nutritional composition and physicochemical characteristics of meat and dairy alternative proteins from four sources: plant proteins, fungal proteins, algal proteins and insect proteins. The difference between these alternative proteins to animal proteins, the effects of their structural features and environmental conditions on their properties, as well as the corresponding mechanism are compared and discussed. Though fungal proteins, algal proteins and insect proteins have shown some advantages over traditional plant proteins, such as the comparable protein content of insect proteins to meat, the better digestibility of fungal proteins and the better foaming properties of algal proteins, there is still a big gap between alternative proteins and meat and dairy proteins. In addition to needing to provide amino acid composition and digestibility similar to animal proteins, alternative proteins also face challenges such as maintaining good solubility and emulsion properties. Their nutritional and physicochemical properties still need thorough investigation, and for commercial application, it is important to develop and optimize industrial technology in alternative protein separation and modification.

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The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) system is a powerful genome editing tool that has been successfully established in some filamentous fungi due to its high flexibility and efficiency. However, the potential toxicity of Cas9 restricts the further popularization and application of this system to some degree. The AMA1 element is a self-replicator derived from Aspergillus nidulans, and its derived vectors can be readily lost without selection. In this study, we eliminated Cas9 toxicity to Fusarium venenatum TB01 based on 100% AMA1-based Cas9 expression vector loss. Meanwhile, two available endogenous Pol III promoters (FvU6374 and Fv5SrRNA) used for sgRNA expression of the CRISPR/Cas9 system were excavated. Compared to FvU6374 (40-50%), Fv5SrRNA exhibited higher single-gene editing efficiency (> 85%), and the efficiency of simultaneous editing of the two genes using Fv5SrRNA was over 75%. Based on this system, a butanediol dehydrogenase encoding gene FvBDH was deleted, and the ethanol yield in variants increased by 52% compared with that of the wild-type. The highly efficient CRISPR/Cas9 system developed here lays the technical foundation for advancing the development of F. venenatum TB01 through metabolic engineering, and the obtained FvBDH gene-edited variants have the potential to simultaneously produce mycoprotein and ethanol by further gene modification and fermentation process optimization in the future.Key points• Cas9 toxicity disappeared and DNA-free gene-edited strains obtained after vector loss• Promoter Fv5SrRNA conferred TB01 higher gene editing efficiency than FvU6374•Deletion of the FvBDH gene resulted in a 52% increase in ethanol yield.

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Excess amount of nitrogen in wastewater has caused serious concerns, such as water eutrophication. Paracoccus pantotrophus MA3, a novel isolated strain of heterotrophic nitrification-anaerobic denitrification bacteria, was evaluated for nitrogen removal using formic acid as the sole carbon source. The results showed that the maximum ammonium removal efficiency was observed under the optimum conditions of 26.25 carbon to nitrogen ratio, 3.39% (v/v) inoculation amount, 34.64 °C temperature, and at 180 rpm shaking speed, respectively. In addition, quantitative real-time PCR technique analysis assured that the gene expression level of formate dehydrogenase, formate tetrahydrofolate ligase, 5,10-methylenetetrahydrofolate dehydrogenase, serine hydroxymethyltransferase, respiratory nitrate reductase beta subunit, L-glutamine synthetase, glutamate dehydrogenase, and glutamate synthase were up-regulated compared to the control group, and combined with nitrogen mass balance analysis to conclude that most of the ammonium was removed by assimilation. A small amount of nitrate and nearly no nitrite were accumulated during heterotrophic nitrification. MA3 exhibited significant denitrification potential under anaerobic conditions with a maximum nitrate removal rate of 4.39 mg/L/h, and the only gas produced was N2. Additionally, 11.50 ± 0.06 mg/L/h of NH4+-N removal rate from biogas slurry was achieved.

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Genetic engineering is one of the most effective methods to obtain fungus strains with desirable traits. However, in some filamentous fungi, targeted gene deletion transformant screening on primary transformation plates is time-consuming and laborious due to a relatively low rate of homologous recombination. A strategy that compensates for the low recombination rate by improving screening efficiency was performed in F. venenatum TB01. In this study, the visualized gene deletion system that could easily distinguish the fluorescent randomly inserted and nonfluorescent putative deletion transformants using green fluorescence protein (GFP) as the marker and a hand-held lamp as the tool was developed. Compared to direct polymerase chain reaction (PCR) screening, the screening efficiency of gene deletion transformants in this system was increased approximately fourfold. The visualized gene deletion system developed here provides a viable method with convenience, high efficiency, and low cost for reaping gene deletion transformants from species with low recombination rates.

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Affected by the rapid population growth, the unbalanced level of social and economic development, the aging population and unhealthy eating patterns, we are facing problems such as lack of food and nutrition, and the high incidence of nutrition related diseases. At the same time, the demand for low-carbon development calls for a sustainable food supply model. Therefore, technologies that meet the taste and nutritional needs of consumers, and serve as a green and sustainable food supply model, such as functional sugar, alternative meat and other future food technologies, have attracted increasing attention. The rapidly developed emerging biomanufacturing technology and its products will support the development of a green and low-carbon future food industry and trigger profound changes in the traditional production mode. Collectively, this represents a major strategic development direction of the emerging bioeconomy. This review summarizes the biomanufacturing technology of functional sugars, microbial proteins and key auxiliary ingredients of alternative meat. We discuss the latest progress in cell factory construction, strain evaluation and process optimization in industrial environment and derived product development. Moreover, future development trend was prospected, with the aim to facilitate industrial development of biomanufacturing of future food.

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With the increase in population growth and environmental pollution, the daily protein supply is facing great challenges. Single-cell protein (SCP) produced by microorganism fermentation is a good alternative for substituting plant- and animal-derived proteins. In this study, Paracoccus communis MA5 isolated from soil previously demonstrated an excellent ability to synthesize SCP directly from sodium formate. To investigate the central metabolic network of formic acid assimilation and protein synthesis, genome-scale analyses were performed. Genomic analysis showed that complete tetrahydrofolate cycle-, serine cycle-, glycolytic pathway-, tricarboxylic acid (TCA) cycle- and nitrogen metabolism-relevant genes were annotated in the genome. These pathways play key roles in the conversion of formic acid into proteins. Transcriptional analysis showed that sodium formate stress could stimulate the metabolic pathway in response to environmental stress, but weaken the sulfur metabolic pathway to inhibit amino acid synthesis, resulting in a decrease in protein content (30% vs 44%). However, under culture conditions with ammonium sulfate, metabolic pathways associated with protein synthesis were accelerated, causing an increase in protein content (53% vs 44%); while the tetrahydrofolate cycle associated with formic acid assimilation was inhibited, causing a 62.5% decrease in growth rate (OD600: 0.21 vs 0.56). These results provide evidence of protein synthesis from sodium formate in strain MA5 at the gene level and lay a theoretical foundation for the optimization of fermentation systems using formic acid as a carbon source.

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Ischemic stroke seriously threatens human health and creates a large social burden. The present study investigated whether tissue inhibitor of metalloproteinases-3 (TIMP3) prevented cerebral ischemia/reperfusion (I/R), with the aim to explore the underlying mechanism. A transient middle cerebral artery occlusion model was conducted in mice, and oxygen glucose deprivation and reoxygenation (OGD/R) was investigated in PC12 cells to mimic cerebral ischemia-reperfusion injury (CIRI). Western blotting was used to determine the expression of TIMP3, Bax, Bcl-2 and AKT. TUNEL was used to detect apoptosis in cerebral tissues or cultured PC12 cells. Expression levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA) were detected to reveal oxidative stress. The results demonstrated that TIMP3 expression was significantly decreased after I/R in vivo or OGD/R in vitro, and the number of TUNEL-positive cells was reduced by the overexpression of TIMP3. The attenuation of Bax/Bcl-2 ratio in OGD/R-induced PC12 cells suppressed the expression levels of ROS and MDA; while also elevating SOD activity in the OGD/R-induced neurocytes in vitro. In addition, TIMP3-overexpression reversed the downregulation of phosphorylated-AKT (Thr308 and Ser473) in OGD/R-treated PC12 cells. However, the anti-apoptotic and anti-oxidative stress roles of TIMP3 in OGD/R-induced PC12 cells were partially abolished after treatment with the AKT inhibitor, AZD5363. Overall, TIMP3 exerted an anti-apoptotic and anti-oxidative stress role in CIRI through the AKT pathway, which may be a potential therapeutic target for the treatment of CIRI.