Increasing energy demands and health-related concerns worldwide have motivated researchers to adopt diverse strategies to improve medium-chain fatty acid (MCFA) biosynthesis for use in the functional food and aviation industries. The abundance of naturally produced MCFAs from botanical sources (i.e., coconut fruit/seeds and palm tree) has been observed to be insufficient compared with the various microorganisms used to cope with industrial demands. Mucor circinelloides is one of many promising microorganisms; it exhibits diverse biotechnological importance ranging from the production of functional lipids to applications in the manufacture of bio-fuel. Thus, research was conducted to acquire the desired elevated amounts of MCFAs (i.e., C8–C12) from metabolically engineered strains of M. circinelloides M65. To achieve this goal, four different acyl-acyl carrier protein (ACP) thioesterase (TE)-encoding genes exhibiting a substrate preference for medium-chain acyl-ACP molecules were expressed in M. circinelloides M65, resulting in the generation of C8–C12 fatty acids. Among all the engineered strains, M65-TE-03 and M65-TE-04 demonstrated the highest production of non-native C8–C10 and C12 fatty acids, respectively, in comparison to the control. These recombinant strains biosynthesized MCFAs de novo within the range from 28 to 46% (i.e., 1.14 to 2.77 g/L) of total cell lipids. Moreover, the reduction in chain length eventually resulted in a 1.5–1.75-fold increase in total lipid productivity in the engineered strains. The MCFAs were also found to be integrated into all lipid classes. This work illustrates how the integration of heterologous enzymes in M. circinelloides can offer a novel opportunity to edit the fatty acid synthases (FAS) complex, resulting in increased production of microbial MFCAs.
Mucor circinelloides has been commonly used as the model microbe to investigate lipid production as an oleaginous fungus. Mitochondrial citrate transporter can catalyze the translocation of the citrate, accumulated from TCA cycle, across the mitochondrial inner membrane. The extra-mitochondrial citrate is then cleaved by ATP-citrate lyase to oxaloacetate (OAA) and acetyl-CoA. Acetyl-CoA together with NADPH generated in cytosol is used for fatty acid biosynthesis. Thus, citrate transporters provide a link between TCA cycle in mitochondria and fatty acid biosynthesis in cytosol. However, the role of citrate transporters for lipid accumulation in oleaginous fungi is not clear. Two genes coding for citrate transporters, named citrate transporter (ct) and tricarboxylate transporter (tct) respectively, were present in the genome of oleaginous fungus M. circinelloides WJ11, a high lipid producing strain (36%, lipid/cell dry weight). As the mutant of strain CBS 277.49 (15%, lipid/cell dry weight) has been constructed and its genetic engineering tools are available for gene manipulation, so in this work, we investigated the role of citrate transporters in regulating lipid biosynthesis by overexpressing the citrate transporters of M. circinelloides WJ11 in CBS 277.49. Results: Our results showed that overexpression of ct and tct led to increased lipid accumulation by 44% (from 13.0% to 18.8%, w/w, CDW) and 68% (from 13.0% to 21.8%, w/w, CDW), respectively. Moreover, extracellular citrate concentration in ct-overexpressing strains (4.91 mM) and tct-overexpressing (3.25 mM) were significantly decreased by 20% and 47% respectively compared to the control (6.09 mM). Furthermore, overexpression of the citrate transporter genes activated the downstream steps in lipid biosynthesis, such as ATP citrate lyase (acl gene) and fatty acid synthases (fas1 and fas2 genes), indicating a greater flux of carbon went into fatty acid biosynthesis. Conclusions: This is the first report showing that citrate transporters involved in lipid accumulation in M. circinelloides. Both citrate transporter and tricarboxylate transporter could transport mitochondrial citrate to cytoplasm, which could provide more citrate to be cleaved by increased ACL to provide more acetyl-CoA and NADPH for increased FAS to synthesize fatty acids, thus, play a vital role in lipid biosynthesis in oleaginous fungus M. circinelloides.
Stearidonic acid (SDA; 18:4, n-3) is the delta 15-desaturase product of gamma linolenic acid (GLA; 18:3, n-6) and delta 6-desaturase product of alpha linolenic acid (ALA; 18:3, n-3). Construction of engineered oleaginous microbes have been attracting significant interest in producing SDA because of its nutritional value and pharmaceutical applications. Mucor circinelloides is a GLA producing filamentous fungus, which can be a useful tool to produce SDA. This study has, therefore, overexpressed the delta-15 desaturase (D15D) gene from Mortierella alpina in this fungus to construct a SDA-producing cell factory. To produce SDA in M. circinelloides, the homologous overexpression of D15D gene was analyzed. When the gene was overexpressed in M. circinelloides CBS 277.49, up to 5.0% SDA was accumulated in this strain. According to current knowledge, this is the first study describing the construction of a SDA-producing cell factory by overexpression of D15D gene in oleaginous fungus M. circinelloides. A new scope for further research has been established by this work to improve SDA production in this fungus, specifically in its high lipid-producing strain, WJ11.
Background Dihomo-gamma linolenic acid (DGLA, 20:3, n-6) is the elongated product of Gamma linolenic acid (GLA, 18:3, n-6) catalyzed by the enzyme delta-6 elongase (D6E) or gamma linolenic acid elongase (GLELO). Construction of engineered oleaginous microbes have been attracting significant interest to produce DGLA because of its nutritional value and medicinal applications. Mucor circinelloides is a GLA producing filamentous fungus which can be a useful tool to produce DGLA. We have, therefore, overexpressed the D6E (GLELO) gene in this fungus to construct DGLA producing cell factory. Result To produce DGLA in M. circinelloides , homologous overexpression of D6E (GLELO) gene was analyzed. When the gene was overexpressed in M. circinelloides CBS277.49, up to 5.72% DGLA was produced in this strain. Conclusion To our knowledge, this is the first report describing the overexpression of D6E (GLELO) gene in M. circinelloides to construct DGLA producing cell factory. A new scope for further research has been established by this work for improved production of DGLA in this fungus, specifically in its high lipid-producing strain, WJ11. Electronic supplementary material The online version of this article (10.1186/s12934-019-1110-4) contains supplementary material, which is available to authorized users.
Autotaxin (ATX) as an important tumor cell motility-stimulating factor is upregulated in many different types of cancer. ATX, a member of the ectonucleotide pyrophosphatase and phosphodiesterase family of enzymes, possesses lysophospholipase D activity which hydrolyzes lysophosphatidylcholine to generate the potent tumor growth factor and mitogen lysophosphatidic acid (LPA). LPA acts on specific G-protein-coupled receptors, thereby regulating cell growth, migration, and survival. This study aimed to investigate the differences in gene expression pattern of ATX between cancerous and adjacent normal tissue of human renal cell carcinoma (RCC) and bladder carcinoma (BC) and find the correlation between ATX expression and clinicopathological features of both of these carcinomas. Both the RCC and BC tissues and with the adjacent normal tissues were collected. Immunohistochemistry and Western blotting analysis were used to detect the extent of ATX expression in all of these samples. Immunohistochemistry and Western blot analysis revealed that expression of ATX protein in carcinoma tissues is significantly higher than that in the adjacent normal tissues. Immunohistochemistry analysis showed that ATX is localized in cytoplasm. Western blotting analysis showed that ATX protein is expressed in both RCC and BC, and the expression levels were 69.5 and 48.0 %, respectively, higher in RCC and BC carcinoma tissue samples than in the adjacent normal tissues, which is consistent with the results of immunohistochemistry study. Thus, this study provided the evidence that ATX is highly expressed in both RCC and BC. Further research can be done to identify the diagnosis and treatment significance of both these carcinomas.
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