Combinatorial Metabolic Engineering for Improving Betulinic Acid Biosynthesis in Saccharomyces cerevisiae.

Betulinic acid (BA) is a lupane-type triterpenoid with potent anticancer and anti-HIV activities. Its great potential in clinical applications necessitates the development of an efficient strategy for BA synthesis. This study attempted to achieve efficient BA biosynthesis in Saccharomyces cerevisiae using systematic metabolic engineering strategies. First, a de novo BA biosynthesis pathway in S. cerevisiae was constructed, which yielded a titer of 14.01 ± 0.21 mg/L. Then, by enhancing the BA synthesis pathway and dynamic inhibition of the competitive pathway, a greater proportion of the metabolic flow was directed toward BA synthesis, achieving a titer of 88.07 ± 5.83 mg/L. Next, acetyl-CoA and NADPH supply was enhanced, which increased the BA titer to 166.43 ± 1.83 mg/L. Finally, another BA synthesis pathway in the peroxisome was constructed. Dual regulation of the peroxisome and cytoplasmic metabolism increased the BA titer to 210.88 ± 4.76 mg/L. Following fed-batch fermentation process modification, the BA titer reached 682.29 ± 8.16 mg/L. Overall, this work offers a guide for building microbial cell factories that are capable of producing terpenoids with efficiency.

Rational Design of Key Enzymes to Efficiently Synthesize Phycocyanobilin in Escherichia coli.

Phycocyanobilin (PCB) is a natural blue tetrapyrrole chromophore that is found in phycocyanin and plays an essential role in photosynthesis. Due to PCB's antioxidation, anti-inflammatory and anti-cancer properties, it has been utilized in the food, pharmaceutical and cosmetic industries. Currently, the extraction of PCB from Spirulina involves complex processes, which has led to increasing interest in the biosynthesis of PCB in Escherichia coli. However, the PCB titer remains low because of the poor activity of key enzymes and the insufficient precursor supply. Here, the synthesis of PCB was firstly improved by screening the optimal heme oxygenase (HO) from Thermosynechococcus elongatus BP-1(HOT) and PCB: ferredoxin oxidoreductase from Synechocystis sp. PCC6803 (PcyAS). In addition, based on a rational design and the infrared fluorescence method for high-throughput screening, the mutants of HOT(F29W/K166D) and PcyAS(D220G/H74M) with significantly higher activities were obtained. Furthermore, a DNA scaffold was applied in the assembly of HOT and PcyAS mutants to reduce the spatial barriers, and the heme supply was enhanced via the moderate overexpression of hemB and hemH, resulting in the highest PCB titer (184.20 mg/L) obtained in a 5 L fermenter. The strategies applied in this study lay the foundation for the industrial production of PCB and its heme derivatives.

The High-Throughput Screening of Microorganisms to Eliminate Ethyl Carbamate in Chinese Liquor.

Ethyl carbamate (EC) is a 2A classified carcinogen in Chinese liquor that has raised many problems regarding food safety. Applying microorganisms to control the content of EC precursors in fermented grains has been proven as an effective method to reduce EC in alcoholic beverages. However, the utilization of microorganisms to decrease the precursors of EC (urea and cyanide) is still incomplete in regard to Chinese liquor. Thus, it is necessary to isolate strains with the degradative activities of urea and cyanide. Herein, Bacillus sonorensis F3 and Bacillus licheniformis YA2 strains were isolated from the fermented grains through multiple rounds of high-throughput screening, and the degradative abilities in urea and cyanide reached 95.72% and 75.48%, respectively. In addition, the urease from the B. sonorensis F3 strain and the carbon nitrogen hydrolase from the B. licheniformis YA2 strain were identified by the heterogeneous expression in Escherichia coli. Then, both F3 and YA2 strains were combined at a ratio of 5:1 and applied to eliminate the EC in the simulated fermentation of Chinese liquor; as a result, 51.10% of EC was reduced without affecting the main composition of flavor substances. The obtained strains have great potential in terms of the improvement of quality and safety of Chinese liquor.

Engineering sulfonate group donor regeneration systems to boost biosynthesis of sulfated compounds.

Sulfonation as one of the most important modification reactions in nature is essential for many biological macromolecules to function. Development of green sulfonate group donor regeneration systems to efficiently sulfonate compounds of interest is always attractive. Here, we design and engineer two different sulfonate group donor regeneration systems to boost the biosynthesis of sulfated compounds. First, we assemble three modules to construct a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) regeneration system and demonstrate its applicability for living cells. After discovering adenosine 5'-phosphosulfate (APS) as another active sulfonate group donor, we engineer a more simplified APS regeneration system that couples specific sulfotransferase. Next, we develop a rapid indicating system for characterizing the activity of APS-mediated sulfotransferase to rapidly screen sulfotransferase variants with increased activity towards APS. Eventually, the active sulfonate group equivalent values of the APS regeneration systems towards trehalose and p-coumaric acid reach 3.26 and 4.03, respectively. The present PAPS and APS regeneration systems are environmentally friendly and applicable for scaling up the biomanufacturing of sulfated products.

Metabolic Engineering of Saccharomyces cerevisiae for Efficient Retinol Synthesis.

Retinol, the main active form of vitamin A, plays a role in maintaining vision, immune function, growth, and development. It also inhibits tumor growth and alleviates anemia. Here, we developed a Saccharomyces cerevisiae strain capable of high retinol production. Firstly, the de novo synthesis pathway of retinol was constructed in S. cerevisiae to realize the production of retinol. Second, through modular optimization of the metabolic network of retinol, the retinol titer was increased from 3.6 to 153.6 mg/L. Then, we used transporter engineering to regulate and promote the accumulation of the intracellular precursor retinal to improve retinol production. Subsequently, we screened and semi-rationally designed the key enzyme retinol dehydrogenase to further increase the retinol titer to 387.4 mg/L. Lastly, we performed two-phase extraction fermentation using olive oil to obtain a final shaking flask retinol titer of 1.2 g/L, the highest titer reported at the shake flask level. This study laid the foundation for the industrial production of retinol.

Is red cell distribution width a prognostic factor in patients with breast cancer? A meta-analysis.

Purpose: The current study aimed to investigate whether red blood cell distribution width (RDW) can predict the prognosis of patients with breast cancer (BC). Methods: We searched four databases, including PubMed, Embase, Cochrane Library databases, and CNKI, from inception to Jun 13, 2022. The primary outcome was overall survival (OS), and the secondary outcome was disease-free survival (DFS). A subgroup analysis was conducted based on different treatments. This meta-analysis was performed with RevMan 5.3 (The Cochrane Collaboration, London, United Kingdom). Results: A total of seven studies including 4,884 BC patients were identified. The high RDW group had a larger tumor size (OR = 2.12, 95% CI = 1.67 to 2.68, P < 0.01), higher proportions of advanced stage tumors (OR = 1.77, 95% CI = 1.38 to 2.27, P < 0.01), more lymph node metastases (OR = 2.00, 95% CI = 1.58 to 2.51, P < 0.01) and lower HER-2 expression (OR = 0.76, 95% CI = 0.61 to 0.95, P = 0.02). For prognosis, after pooling all the data, we found that the high RDW group was associated with worse OS (HR = 2.12, 95% CI = 1.47 to 3.08, P < 0.01) and DFS (HR = 1.77, 95% CI = 1.32 to 2.37, P < 0.01). The subgroup analysis found that RDW had prognostic significance but only for surgery-only patients (HR = 2.41, 95% CI = 1.67 to 3.49, P < 0.01). Conclusion: High RDW was associated with worse OS and DFS. Therefore, RDW was a simple predictive factor for the prognosis of BC patients.

Combinatorial Protein Engineering and Metabolic Engineering for Efficient Synthesis of l-Histidine in Corynebacterium glutamicum.

l-Histidine is an essential proteinogenic amino acid in food with extensive applications in the pharmaceutical field. Herein, we constructed a Corynebacterium glutamicum recombinant strain for efficient biosynthesis of l-histidine. First, to alleviate the l-histidine feedback inhibition, the ATP phosphoribosyltransferase mutant HisGT235P-Y56M was constructed based on molecular docking and high-throughput screening, resulting in the accumulation of 0.83 g/L of l-histidine. Next, we overexpressed rate-limiting enzymes including HisGT235P-Y56M and PRPP synthetase and knocked out the pgi gene in the competing pathway, which increased the l-histidine production to 1.21 g/L. Furthermore, the energy status was optimized by decreasing the reactive oxygen species level and enhancing the supply of adenosine triphosphate, reaching a titer of 3.10 g/L in a shake flask. The final recombinant strain produced 5.07 g/L of l-histidine in a 3 L bioreactor, without the addition of antibiotics and chemical inducers. Overall, this study developed an efficient cell factory for l-histidine biosynthesis by combinatorial protein engineering and metabolic engineering.

Construction of a Protein Crystalline Inclusion-Based Enzyme Immobilization System for Biosynthesis of PAPS from ATP and Sulfate.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is the bioactive form of sulfate and is involved in all biological sulfation reactions. The enzymatic transformation method for PAPS is promising, but the low efficiency and high cost of enzyme purification and storage restrict its practical applications. Here, we reported PAPS biosynthesis with a protein crystalline inclusion (PCI)-based enzyme immobilization system. First, the in vivo crystalline inclusion protein CipA was identified as an efficient auto-assembly tag for immobilizing the bifunctional PAPS synthase (ASAK). After characterizing the pyrophosphokinase activity of a polyphosphate exonuclease PaPPX from Pseudomonas aeruginosa, and optimizing the linker fragment, auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA were constructed. Then, the auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA with high stability were co-expressed and immobilized for constructing a transformation system. The highest transformation rate of PAPS from ATP and sulfate reached 90%, and the immobilized enzyme can be reused 10 times. The present work provided a convenient, efficient, and easy to be enlarged auto-immobilization system for PAPS biosynthesis from ATP and sulfate. The immobilization system also represented a new approach to reduce the production cost of PAPS by facilitating the purification, storage, and reuse of related enzymes, and it would boost the studies on biotechnological production of glycosaminoglycans and sulfur-containing natural compounds.

ATP-Binding Cassette Exporter PDR11-Mediated Terpenoid Secretion in Engineered Yeast.

The metabolic burden caused by terpenoid accumulation limits the development of highly efficient microbial cell factories, which can be circumvented using exporter-mediated product secretion. Although our previous study showed that the pleiotropic drug resistance exporter (PDR11) mediates the export of rubusoside in Saccharomyces cerevisiae, the underlying mechanism is still unclear. Herein, we used GROMACS software to simulate PDR11-mediated rubusoside recruitment and found six residues (D116, D167, Y168, P521, R663, and L1146) on PDR11 that are critical for this process. We also explored the exportation potential of PDR11 for 39 terpenoids by calculating their binding affinity using batch molecular docking. Then, we verified the accuracy of the predicted results by conducting experiments with squalene, lycopene, and ß-carotene as examples. We found that PDR11 can efficiently secrete terpenoids with binding affinities lower than -9.0 kcal/mol. Combining the computer-based prediction and experimental verification, we proved that binding affinity is a reliable parameter to screen exporter substrates and might potentially enable rapid screening of exporters for natural products in microbial cell factories.

Combinatorial metabolic engineering enables the efficient production of ursolic acid and oleanolic acid in Saccharomyces cerevisiae.

Ursolic acid (UA) and oleanolic acid (OA) have been demonstrated to have promising therapeutic potential as anticancer and bacteriostasis agents. Herein, via the heterologous expression and optimization of CrAS, CrAO, and AtCPR1, the de novo syntheses of UA and OA were achieved with titers of 7.4 and 3.0 mg/L, respectively. Subsequently, metabolic flux was redirected by increasing the cytosolic acetyl-CoA level and tuning the copy numbers of ERG1 and CrAS, thereby affording 483.4 mg/L UA and 163.8 mg/L OA. Furthermore, the lipid droplet compartmentalization of CrAO and AtCPR1 alongside the strengthening of the NADPH regeneration system increased the UA and OA titers to 692.3 and 253.4 mg/L in a shake flask and to 1132.9 and 433.9 mg/L in a 3-L fermenter, which is the highest UA titer reported to date. Overall, this study provides a reference for constructing microbial cell factories that can efficiently synthesize terpenoids.

Simulation and Control of the Formation of Ethyl Carbamate during the Fermentation and Distillation Processes of Chinese Baijiu.

Baijiu is a popular alcoholic beverage with a long history in China. However, the widespread presence of the ethyl carbamate (EC) carcinogen has raised many food safety concerns. To date, the main precursors of EC and its formation process have not been determined, resulting in difficulty controlling EC in Baijiu. In this study, the main precursors of EC are identified as urea and cyanide during the process of brewing for different flavors of Baijiu, while the dominant stage in which EC formation occurs is during the process of distillation rather than fermentation. In addition, the effects of temperature, pH value, alcohol concentration and metal ions on the formation of EC are confirmed. In the following study, the main precursor of EC is identified as cyanide during the process of distillation, and a combination of optimizing the distillation device and adding copper wire is proposed. Furthermore, the effect of this novel strategy is examined in gaseous reactions between cyanide and ethanol, reducing the concentration of EC by 74.0%. Finally, the feasibility of this strategy is verified in simulated distillations of fermented grains, reducing the formation of EC by 33.7-50.2%. This strategy has great application potential in industrial production.

Moromi mash dysbiosis trigged by salt reduction is relevant to quality and aroma changes of soy sauce.

Health concerns related to excessive salt consumption have increased the demand for foods with reduced salt content. However, it is a challenge to perform low salt fermentation of high salt liquid-state (HSL) soy sauce due to the interplay between salt and microorganisms. In this study, ≤12 % (w/v) NaCl led to failed fermentation of HSL soy sauce. At 9 % (w/v) NaCl, amino acid nitrogen decreased to 0.31 g/100 mL, total acid increased to 10.1 g/L, and biogenic amines increased to 904.49 mg/L. With reduced salt, the total number of bacteria (1-2 orders of magnitude) and spoilage bacteria (Bacillus, Kurthia, Staphylococcus saprophyticus, and Lactobacillus pobuzihii) increased, and the total number of functional microorganisms (Weissella, Zygosaccharomyces, and Candida) decreased. Unacceptable volatiles contents were higher in reduced-salt soy sauce than in normal salt soy sauce. Most of the unacceptable volatiles were positively correlated with spoilage bacteria.

Naringenin Promotes Myotube Formation and Maturation for Cultured Meat Production.

Cultured meat is an emerging technology for manufacturing meat through cell culture rather than animal rearing. Under most existing culture systems, the content and maturity of in vitro generated myotubes are insufficient, limiting the application and public acceptance of cultured meat. Here we demonstrated that a natural compound, naringenin (NAR), promoted myogenic differentiation of porcine satellite cells (PSCs) in vitro and increased the content and maturity of generated myotubes, especially for PSCs that had undergone extensive expansion. Mechanistically, NAR upregulated the IGF-1/AKT/mTOR anabolic pathway during the myogenesis of PSCs by activating the estrogen receptor ß. Moreover, PSCs were mixed with hydrogels and cultured in a mold with parallel micro-channels to manufacture cultured pork samples. More mature myosin was detected, and obvious sarcomere was observed when the differentiation medium was supplemented with NAR. Taken together, these findings suggested that NAR induced the differentiation of PSCs and generation of mature myotubes through upregulation of the IGF-1 signaling, contributing to the development of efficient and innovative cultured meat production systems.

Profiling the role of microorganisms in quality improvement of the aged flue-cured tobacco.

BACKGROUND: The aging process in the tobacco production, as in other food industries, is an important process for improving the quality of raw materials. In the spontaneous aging, the complex components in flue-cured tobacco (FT) improve flavor or reduce harmful compounds through chemical reactions, microbial metabolism, and enzymatic catalysis. Some believed that tobacco-microbe played a significant part in this process. However, little information is available on how microbes mediate chemical composition to improve the quality of FT, which will lay the foundation for the time-consuming spontaneous aging to seek ways to shorten the aging cycle. RESULTS: Comparing aged and unaged FT, volatile and non-volatile differential compounds (DCs) were multi-dimensionally analyzed with the non-targeted metabolomes based on UPLC-QTOP-MS (the ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry), GC-MS (gas chromatography-mass spectrometer) assisted derivatization and HP-SPME-GC/MS (headspace solid-phase micro-extraction assisted GC-MS). Products associated with the degradation pathways of terpenoids or higher fatty acids were one of the most important factors in improving FT quality. With the microbiome, the diversity and functions of microbial flora were analyzed. The high relative abundance function categories were in coincidence with DCs-related metabolic pathways. According to the correlation analysis, Acinetobacter, Sphingomonas and Aspergillus were presumed to be the important contributor, in which Aspergillus was associated with the highest number of degradation products of terpenoids and higher fatty acids. At last, the screened Aspergillus nidulans strain F4 could promote the degradation of terpenoids and higher fatty acids to enhance tobacco flavor by secreting highly active lipoxygenase and peroxidase, which verified the effect of tobacco-microbes on FT quality. CONCLUSIONS: By integrating the microbiome and metabolome, tobacco-microbe can mediate flavor-related substances to improve the quality of FT after aging, which provided a basis for identifying functional microorganisms for reforming the traditional spontaneous aging.

Enzymatic Production of Chondroitin Oligosaccharides and Its Sulfate Derivatives.

Chondroitin sulfate (CS) has a wide range of physiological functions and clinical applications. However, the biosynthesis of chondroitin oligosaccharides (o-CHs) and sulfate derivatives with specific length is always challenging. Herein, we report enzymatic strategies for producing homogeneous o-CHs and its sulfate derivatives from microbial sourced chondroitin. Chondroitin disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, and decasaccharides with defined structure were produced by controllably depolymerizing microbial sourced chondroitin with an engineered chondroitinase ABC I. The highest conversion rates of the above corresponding o-CHs were 65.5%, 32.1%, 12.7%, 7.2%, and 16.3%, respectively. A new efficient enzymatic sulfation system that directly initiates from adenosine 5'-triphosphate (ATP) and sulfate was developed and improved the sulfation of chondroitin from 8.3% to 85.8% by optimizing the temperature, sulfate and ATP concentration. o-CHs decasaccharide, octasaccharide, hexasaccharide, tetrasaccharide and disaccharide were modified and the corresponding sulfate derivatives with one sulfate group were prepared. The enzymatic approaches constructed here for preparing o-CHs and its sulfate derivatives pave the way for the study of structure-activity relationship and applications.

Effects of Inoculation With Acinetobacter on Fermentation of Cigar Tobacco Leaves.

Metabolic activity of the microbial community greatly affects the quality of cigar tobacco leaves (CTLs). To improve the quality of CTLs, two extrinsic microbes (Acinetobacter sp. 1H8 and Acinetobacter indicus 3B2) were inoculated into CTLs. The quality of CTLs were significantly improved after fermentation. The content of solanone, 6-methyl-5-hepten-2-one, benzeneacetic acid, ethyl ester, cyclohexanone, octanal, acetophenone, and 3,5,5-trimethyl-2-cyclohexen-1-one were significantly increased after inoculated Acinetobacter sp. 1H8. The inoculation of Acinetobacter sp. 1H8 enhanced the normal evolutionary trend of bacterial community. The content of trimethyl-pyrazine, 2,6-dimethyl-pyrazine, and megastigmatrienone were significantly increased after inoculated Acinetobacter indicus 3B2. The inoculation of Acinetobacter indicus 3B2 completely changed the original bacterial community. Network analysis revealed that Acinetobacter was negatively correlated with Aquabacterium, positively correlated with Bacillus, and had significant correlations with many volatile flavor compounds. This work may be helpful for improving fermentation product quality by regulating microbial community, and gain insight into the microbial ecosystem.

Analysis of Microbial Community, Volatile Flavor Compounds, and Flavor of Cigar Tobacco Leaves From Different Regions.

Despite the booming international trade in cigar tobacco leaves (CTLs), the main characteristics of tobacco leaves from different producing areas are rarely reported. This study aimed to characterize the microbial community, volatile flavor compounds (VFCs), and flavor of CTLs from four famous cigar-producing areas, including Dominica, Brazil, Indonesia, and China. High-throughput sequencing results showed that the dominant genera in CTLs were Staphylococcus, Pseudomonas, Aspergillus, Sampaiozyma, and Alternaria. Sensory analysis revealed that Indonesian and Chinese CTLs were characterized by leathery, peppery, and baked aroma. Brazilian CTLs were dominated by caramel and herb aroma. Dominican CTLs had aromas of milk, fruity, sour, cream, flower, nutty, and honey. Supplemented with the determination of volatile flavor compounds (VFCs), the flavor of CTLs could be scientifically quantified. Most of these VFCs were aldehydes and ketones, and 20 VFCs showed significant differences in CTLs from different regions. The microbial community, VFCs, and flavor of CTLs vary widely due to geographic differences. Network analysis revealed the microbial community was closely related to most VFCs, but the relationships between the fungal community and VFCs were less than the bacterial community, and most of them were negative. Furthermore, it also found that the bacterial community had a greater contribution to the flavor of CTLs than the fungal community. This study obtained essential information on CTLs, which laid a foundation for deeply excavating the relationship between microbes and VFCs and flavor, and establishing a tobacco information database.

Metabolite-based cell sorting workflow for identifying microbes producing carbonyls in tobacco leaves.

Carbonyl compounds represented by aldehydes and ketones make an important contribution to the flavor of tobacco. Since most carbonyl compounds are produced by microbes during tobacco fermentation, identifying their producers is important to improve the quality of tobacco. Here, we created an efficient workflow that combines metabolite labeling with fluorescence-activated cell sorting (ML-FACS), 16S rRNA gene sequencing, and microbial culture to identify the microbes that produce aldehydes or ketones in fermented cigar tobacco leaves (FCTL). Microbes were labeled with a specific fluorescent dye (cyanine5 hydrazide) and separated by flow cytometry. Subsequently, the sorted microbes were identified and cultured under laboratory conditions. Four genera, Acinetobacter, Sphingomonas, Solibacillus, and Lysinibacillus, were identified as the main carbonyl compound-producing microbes in FCTL. In addition, these microorganisms could produce flavor-related aldehydes and ketones in a simple synthetic medium, such as benzaldehyde, phenylacetaldehyde, 4-hydroxy-3-ethoxy-benzaldehyde, and 3,5,5-trimethyl-2-cyclohexene-1-one. On the whole, this research has developed a new method to quickly isolate and identify microorganisms that produce aldehydes or ketones from complex microbial communities. ML-FACS would also be used to identify other compound-producing microorganisms in other systems. KEY POINTS: • An approach was developed to identify target microbes in complex communities. • Microbes that produce aldehyde/ketone flavor compounds in fermented cigar tobacco leaves were identified. • Functional microbes that produce aldehyde/ketone flavor compounds from the native environment were captured in pure cultures.

Correlation between the microbial community and ethyl carbamate generated during Huzhou rice wine fermentation.

Ethyl carbamate (EC) is a potential carcinogen that is mainly produced by the spontaneous reaction between urea and ethanol during rice wine brewing. Huzhou rice wine (HZRW) is a traditional Chinese rice wine, but the correlation between its urea content and the microbial communities present during the fermentation process has not yet been evaluated. In this study, high-throughput sequencing technology was used to monitor the microbial community composition of HZRW in the different fermentation stages. The correlations between the microbial community and the physical and chemical properties and EC, urea and arginine contents were evaluated using the redundancy analysis (RDA) method. The metabolic profiles of key genes in the arginine and urea metabolic pathways were obtained via phylogenetic investigation of the communities by reconstruction of unobserved states (PICRUSt). The results showed that the fungal genera Saccharomyces, Issatchenkia, Torulaspora and Rhizopus were dominant during the fermentation of HZRW. Weissella and Acinetobacter were the dominant bacterial genera in the early stage, while Weissella, Staphylococcus, Leuconostoc and Streptophyta were the dominant bacterial genera in the late stage. Urea and arginine were positively correlated with Saccharomyces, Lactobacillus and Staphylococcus. In addition, the dominant genera of both fungi and bacteria were involved in the metabolism of arginine and urea. Finally, the relationships between the dominant microorganisms and key genes of the arginine and urea metabolic pathways were established. The obtained results are helpful in better understanding the mechanisms of metabolism of arginine and urea during rice wine fermentation and therefore improving the safety profile of rice wine.

IGF-1 Signaling Regulates Mitochondrial Remodeling during Myogenic Differentiation.

Skeletal muscle is essential for locomotion, metabolism, and protein homeostasis in the body. Mitochondria have been considered as a key target to regulate metabolic switch during myo-genesis. The insulin-like growth factor 1 (IGF-1) signaling through the AKT/mammalian target of rapamycin (mTOR) pathway has a well-documented role in promoting muscle growth and regeneration, but whether it is involved in mitochondrial behavior and function remains un-examined. In this study, we investigated the effect of IGF-1 signaling on mitochondrial remodeling during myogenic differentiation. The results demonstrated that IGF-1 signaling stimulated mitochondrial biogenesis by increasing mitochondrial DNA copy number and expression of genes such as Cox7a1, Tfb1m, and Ppargc1a. Moreover, the level of mitophagy in differentiating myoblasts elevated significantly with IGF-1 treatment, which contributed to mitochondrial turnover. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) were identified as two key mediators of IGF-1-induced mitochondrial biogenesis and mitophagy, respectively. In addition, IGF-1 supplementation could alleviate impaired myoblast differentiation caused by mitophagy deficiency, as evidenced by increased fusion index and myosin heavy chain expression. These findings provide new insights into the role of IGF-1 signaling and suggest that IGF-1 signaling can serve as a target for the research and development of drugs and nutrients that support muscle growth and regeneration.