Universal and unique strategies for the production of polyunsaturated fatty acids in industrial oleaginous microorganisms.

Polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (ARA), are beneficial for reducing blood cholesterol and enhancing memory. Traditional PUFA production relies on extraction from plants and animals, which is unsustainable. Thus, using microorganisms as lipid-producing factories holds promise as an alternative way for PUFA production. Several oleaginous microorganisms have been successfully industrialized to date. These can be divided into universal and specialized hosts according to the products range of biosynthesis. The Yarrowia lipolytica is universal oleaginous host that has been engineered to produce a variety of fatty acids, such as γ-linolenic acid (GLA), EPA, ARA and so on. By contrast, the specialized host are used to produce only certain fatty acids, such as ARA in Mortierella alpina, EPA in Nannochloropsis, and DHA in Thraustochytrids. The metabolic engineering and fermentation strategies for improving PUFA production in universal and specialized hosts are different, which is the subject of this review. In addition, the widely applicable strategies for microbial lipid production that are not specific to individual hosts were also reviewed.

Identification of lipid synthesis genes in Schizochytrium sp. and their application in improving eicosapentaenoic acid synthesis in Yarrowia lipolytica.

BACKGROUND: Eicosapentaenoic acid (EPA) is widely used in the functional food and nutraceutical industries due to its important benefits to human health. Oleaginous microorganisms are considered a promising alternative resource for the production of EPA lipids. However, the storage of EPA in triglyceride (TG) becomes a key factor limiting its level. RESULTS: This study aimed to incorporate more EPA into TG storage through metabolic engineering. Firstly, key enzymes for TG synthesis, the diacylglycerol acyltransferase (DGAT) and glycerol-3-phosphate acyltransferase (GPAT) genes from Schizochytrium sp. HX-308 were expressed in Yarrowia lipolytica to enhance lipid and EPA accumulation. In addition, engineering the enzyme activity of DGATs through protein engineering was found to be effective in enhancing lipid synthesis by replacing the conserved motifs "HFS" in ScDGAT2A and "FFG" in ScDGAT2B with the motif "YFP". Notably, combined with lipidomic analysis, the expression of ScDGAT2C and GPAT2 enhanced the storage of EPA in TG. Finally, the accumulation of lipid and EPA was further promoted by identifying and continuing to introduce the ScACC, ScACS, ScPDC, and ScG6PD genes from Schizochytrium sp., and the lipid and EPA titer of the final engineered strain reached 2.25 ± 0.03 g/L and 266.44 ± 5.74 mg/L, respectively, which increased by 174.39% (0.82 ± 0.02 g/L) and 282.27% (69.70 ± 0.80 mg/L) compared to the initial strain, respectively. CONCLUSION: This study shows that the expression of lipid synthesis genes from Schizochytrium sp. in Y. lipolytica effectively improves the synthesis of lipids and EPA, which provided a promising target for EPA-enriched microbial oil production.

Amlodipine inhibits the proliferation and migration of esophageal carcinoma cells through the induction of endoplasmic reticulum stress.

BACKGROUND: L-type calcium channels are the only protein channels sensitive to calcium channel blockers, and are expressed in various cancer types. The Cancer Genome Atlas database shows that the mRNA levels of multiple L-type calcium channel subunits in esophageal squamous cell carcinoma tumor tissue are significantly higher than those in normal esophageal epithelial tissue. Therefore, we hypothesized that amlodipine, a long-acting dihydropyridine L-type calcium channel blocker, may inhibit the occurrence and development of esophageal cancer (EC). AIM: To investigate the inhibitory effects of amlodipine on EC through endoplasmic reticulum (ER) stress. METHODS: Cav1.3 protein expression levels in 50 pairs of EC tissues and corresponding paracancerous tissues were examined. Subsequently, the inhibitory effects of amlodipine on proliferation and migration of EC cells in vitro were detected using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and Transwell assays. In vivo experiments were performed using murine xenograft model. To elucidate the underlying mechanisms, in vitro cell studies were performed to confirm that ER stress plays a role in inhibition proliferation and migration of EC cells treated with amlodipine. RESULTS: The expression level of Cav1.3 in esophageal carcinoma was 1.6 times higher than that in paracancerous tissues. Amlodipine treatment decreased the viability of esophageal carcinoma cells in a dose- and time-dependent manner. In vivo animal experiments also clearly indicated that amlodipine inhibited the growth of EC tumors in mice. Additionally, amlodipine reduces the migration of tumor cells by inhibiting epithelial-mesenchymal transition (EMT). Mechanistic studies have demonstrated that amlodipine induces ER stress-mediated apoptosis and suppresses EMT. Moreover, amlodipine-induced autophagy was characterized by an increase in autophagy lysosomes and the accumulation of light chain 3B protein. The combination of amlodipine with the ER stress inhibitor 4-phenylbutyric acid further confirmed the role of the ER stress response in amlodipine-induced apoptosis, EMT, and autophagy. Furthermore, blocking autophagy increases the ratio of apoptosis and migration. CONCLUSION: Collectively, we demonstrate for the first time that amlodipine promotes apoptosis, induces autophagy, and inhibits migration through ER stress, thereby exerting anti-tumor effects in EC.

Enhancing the accumulation of lipid and docosahexaenoic acid in Schizochytrium sp. by co-overexpression of phosphopantetheinyl transferase and ω-3 fatty acid desaturase.

Docosahexaenoic acid (DHA) as one of ω-3 polyunsaturated fatty acids (PUFAs), plays a key role in brain development, and is widely used in food additives and the pharmaceutical industry. Schizochytrium sp. is often considered as a satisfactory strain for DHA industrialization. The aim of this study was to assess the feasibility of phosphopantetheinyl transferase (PPTase) and ω-3 fatty acid desaturase (FAD) for regulating DHA content in Schizochytrium sp. PPTase is essential to activate the polyketide-like synthase (PKS) pathway, which can transfer apo-acyl-carrier protein (apo-ACP) into holo-ACP, and plays a key role in DHA synthesis. Moreover, DHA and docosapentaenoic acid (DPA) are synthesized by the PKS pathway simultaneously, so high DPA synthesis limits the increase of DHA content. In addition, the detailed mechanisms of PKS pathway have not been fully elucidated, so it is difficult to improve DHA content by modifying PKS. However, ω-3 FAD can convert DPA into DHA, and it is the most direct and effective way to increase DHA content and reduce DPA content. Based on this, PPTase was overexpressed to enhance the synthesis of DHA by the PKS pathway, overexpressed ω-3 FAD to convert the co-product of the PKS pathway into DHA, and co-overexpressed PPTase and ω-3 FAD. With these strategies, compared with wild type, the final lipid, and DHA titer were 92.5 and 51.5 g L-1 , which increased by 46.4% and 78.1%, respectively. This study established an efficient DHA production strain, and provided some feasible strategies for industrial DHA production in Schizochytrium sp.

Enhancing the biomass and docosahexaenoic acid-rich lipid accumulation of Schizochytrium sp. in propionate wastewater.

In order to find a more effective way to obtain docosahexaenoic acid (DHA) rich lipid from Schizochytrium sp., a widespread propionate wastewater (PW) is used. PW is a common industrial and domestic wastewater, and transforming it into valuable products is a potential treatment method. Schizochytrium sp. is a rapidly growing oleaginous organism, which has been used commercially for DHA production. Herein, PW is completely used for DHA production by Schizochytrium sp. by genetic engineering and fermentation optimization, which can alleviate the increasingly tense demand for water resources and environmental pollution caused by industrial wastewater. Firstly, the methylmalonyl-CoA mutase (MCM) was overexpressed in Schizochytrium sp. to enhance the metabolism of propionate, then the engineered strain of overexpressed MCM (OMCM) can effectively use propionate. Then, the effects of PW with different concentration of propionate were investigated, and results showed that OMCM can completely replace clean water with PW containing 5 g L-1 propionate. Furthermore, in the fed-batch fermentation, the OMCM obtained the highest biomass of 113.4 g L-1 and lipid yield of 64.4 g L-1 in PW condition, which is 26.8% and 51.7% higher than that of wild type (WT) in PW condition. Moreover, to verify why overexpression of MCM can promote DHA and lipid accumulation, the comparative metabolomics, ATP production level, the antioxidant system, and the transcription of key genes were investigated. Results showed that ATP induced by PW condition could drive the synthesis of DHA, and remarkably improve the antioxidant capacity of cells by enhancing the carotenoids production. Therefore, PW can be used as an effective and economical substrate and water source for Schizochytrium sp. to accumulate biomass and DHA.

Efficient Biosynthesis of Odd-Chain Fatty Acids via Regulating the Supply and Consumption of Propionyl-CoA in Schizochytrium sp.

Odd chain fatty acids (OCFAs) are high-value-added compounds with great application in the field of food and medicine. As an oleaginous microorganism, Schizochytrium sp. has the potential to produce OCFAs efficiently. Propionyl-CoA is used as a precursor to synthesize OCFAs through the fatty acid synthetase (FAS) pathway, so its flow direction determines the yield of OCFAs. Here, different substrates were assessed to promote propionyl-CoA supply for OCFA accumulation. Moreover, the methylmalonyl-CoA mutase (MCM) was identified as the key gene responsible for propionyl-CoA consumption, which promotes the propionyl-CoA to enter into the tricarboxylic acid cycle rather than the FAS pathway. As one of the classic B12-dependent enzymes, the activity of MCM can be inhibited in the absence of B12. As expected, the OCFA accumulation was greatly increased. However, the removal of B12 caused growth limitation. Furthermore, the MCM was knocked out to block the consumption of propionyl-CoA and to maintain cell growth; results showed that the engineered strain achieved the OCFAs titer of 2.82 g/L, which is 5.76-fold that of wild type. Last, a fed-batch co-feeding strategy was developed, resulting in the highest reported OCFAs titer of 6.82 g/L. This study provides guidance for the microbial production of OCFAs.

[Effects of aerobic exercise on Nrf2/GPX4/Ferroptosis pathway in myocardial injury in high-fat diet mice].

Objective: To illuminate the protective effects of pathway in inhibiting ferroptosis by glutathione peroxidase 4 (GPX4) activated by nuclear factor erythroid 2-related factor 2 (Nrf2) during aerobic exercise against myocardial injury in high-fat diet mice. Methods: Forty 5-week-old SPF C57BL/6 male mice were randomly divided into the control group (NC), the exercise group (NE), the high fat group (HC) and the high fat diet with exercise group (HE, began at the same time). There were 10 mice in each group. The mice in the high fat diet group were fed with 60% Kcal SPF high fat model diet. Aerobic exercise was performed using increasing load platform exercise, 5 days /week, 60 min/d, the speed started from 13m/min, and increased by 1m/min every two weeks. Myocardium and blood samples were collected after 14 weeks. Structural changes of myocardial tissues were observed by HE staining. Western blot was used to detect the expressions of Nrf2/GPX4/Ferroptosis related proteins in myocardium. Myocardial peroxide concentration and antioxidant enzyme activity were measured by spectrophotometry. Myocardial mitochondrial 8-hydroxy-2 deoxyguanosine (8-OHdG) and serum insulin were measured by ELISA. Results: Compared with the NC group, there was more lipid accumulation in the myocardial fiber space in the HC group, and the levels of FBG and FINS were increased significantly, while ISI was decreased significantly (P＜0.01). Compared with the HC group, the lipid concentration was decreased in the HE group, and the activities of total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD) and glutathione (GSH) were increased significantly, while the levels of mitochondrial 8-OHdG and myocardial iron content were decreased (P＜0.01). The expression levels of Ferroportin1 (FPN1), ferritin heavy chain 1 (FTH1), GPX4, glucose transporter (GLUT1) and Nrf2 in the HE group were significantly higher than those in the HC group (P＜0.01). Conclusion: The expression of GPX4 was enhanced by more Nrf2 transposition into the nuclear during aerobic exercise, which inhibited the occurrence of myocardial ferroptosis. The activities of antioxidant enzymes were promoted and inhibited the peroxidation damage of myocardial mitochondria.

Inhibition of bromodomain-containing protein 4 enhances the migration of esophageal squamous cell carcinoma cells by inducing cell autophagy.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC), the predominant type of esophageal cancer, has a 5-year survival rate less than 20%. Although the cause of poor prognosis is the high incidence and mortality of ESCC, the high rate of metastasis after esophageal cancer surgery is the main cause of death after the surgery. Bromodomain-containing protein 4 (BRD4), an epigenetic reader of chromatin-acetylated histones in tumorigenesis and development, plays an essential role in regulating oncogene expression. BRD4 inhibition and BRD4 inhibition-based treatment can potentially suppress ESCC growth. However, the effects and mechanisms of action of BRD4 on ESCC cell migration remain unclear. AIM: To explore the effect of BRD4 on cell migration of ESCC in vitro and its possible molecular mechanism. METHODS: Human ESCC cell lines KYSE-450 and KYSE-150 were used. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was performed to examine cell proliferation, and the transwell migration assay was conducted to test ESCC cell migration. JQ1, a BRD4 inhibitor, was applied to cells, and BRD4 siRNA was transfected into ESCC cells to knockdown endogenous BRD4. GFP-RFP-LC3 adenovirus was infected into ESCC cells to evaluate the effect of JQ1 on autophagy. Western blotting was performed to determine the protein levels of BRD4, E-cadherin, vimentin, AMP-activated protein kinase (AMPK), and p-AMPK. RESULTS: BRD4 was either downregulated by small interfering RNA or pretreated with JQ1 in ESCC cells, leading to increased tumor migration in ESCC cells in a dose- and time-dependent manner. Inhibition of BRD4 not only significantly suppressed cell proliferation but also strongly increased cell migration by inducing epithelial-mesenchymal transition (EMT). The protein expression of vimentin was increased and E-cadherin decreased in a dose-dependent manner, subsequently promoting autophagy in KYSE-450 and KYSE-150 cells. Pretreatment with JQ1, a BRD4 inhibitor, inhibited BRD4-induced LC3-II activation and upregulated AMPK phosphorylation in a dose-dependent manner. Additionally, an increased number of autophagosomes and autolysosomes were observed in JQ1-treated ESCC cells. The autophagy inhibitor 3-methyladenine (3-MA) reversed the effects of BRD4 knockdown on ESCC cell migration and blocked JQ1-induced cell migration. 3-MA also downregulated the expression of vimentin and upregulation E-cadherin. CONCLUSION: BRD4 inhibition enhances cell migration by inducing EMT and autophagy in ESCC cells via the AMPK-modified pathway. Thus, the facilitating role on ESCC cell migration should be considered for BRD4 inhibitor clinical application to ESCC patients.

Antibacterial and Antifungal Sesquiterpenoids: Chemistry, Resource, and Activity.

Infectious diseases caused by bacteria and fungi are threatening human health all over the world. It is an increasingly serious problem that the efficacies of some antibacterial and antifungal agents have been weakened by the drug resistance of some bacteria and fungi, which makes a great need for new antibiotics. Sesquiterpenoids, with abundant structural skeleton types and a wide range of bioactivities, are considered as good candidates to be antibacterial and antifungal agents. In the past decades, many sesquiterpenoids were isolated from plants and fungi that exhibited good antibacterial and antifungal activities. In this review, the names, source, structures, antibacterial and antifungal degrees, and mechanisms of sesquiterpenoids with antibacterial and antifungal activity from 2012 to 2022 are summarized, and the structure-activity relationship of these sesquiterpenoids against bacteria and fungi is also discussed.

Feasibility of MRI Radiomics for Predicting KRAS Mutation in Rectal Cancer.

The mutation status of KRAS is a significant biomarker in the prognosis of rectal cancer. This study investigated the feasibility of MRI-based radiomics in predicting the mutation status of KRAS with a composite index which could be an important criterion for KRAS mutation in clinical practice. In this retrospective study, a total of 127 patients with rectal cancer were enrolled. The 3D Slicer was used to extract the radiomics features from the MRI images, and sparse support vector machine (SVM) with linear kernel was applied for feature reduction. The radiomics classifier for predicting the KRAS status was then constructed by Linear Discriminant Analysis (LDA) and its performance was evaluated. The composite index was determined with LDA model. Out of 127 rectal cancer subjects, there were 44 KRAS mutation cases and 83 wild cases. A total of 104 radiomics features were extracted, 54 features were filtered by linear SVM with L1-norm regularization and 6 features that had no significant correlations within them were finally selected. The radiomics classifier constructed using the 6 features featured an AUC value of 0.669 (specificity: 0.506; sensitivity: 0.773) with LDA. Furthermore, the composite index (Radscore) had statistically significant difference between the KRAS mutation and wild groups. It is suggested that the MRI-based radiomics has the potential in predicting the KRAS status in patients with rectal cancer, which may enhance the diagnostic value of MRI in rectal cancer.