γ-Linolenic acid (GLA) is important because of its nutritional value and medicinal applications. Although the biosynthetic pathways of some plant and microbial GLA have been deciphered, current understanding of the correlation between desaturases and GLA synthesis in oleaginous fungi is incomplete. In previous work, we found that a large amount of oleic acid (OA) had not been converted to linoleic acid (LA) or GLA in Mucor circinelloides CBS 277.49, which may be due to inadequate activities of the delta-12 or delta-6 desaturases, and thus leading to the accumulation of OA and LA. Thus, it is necessary to explore the main contributing factor during the process of GLA biosynthesis in M. circinelloides. To enhance GLA production in M. circinelloides, homologous overexpression of delta-12 and two delta-6 desaturases (named delta-6-1 and delta-6-2, respectively) were analyzed. When delta-6 desaturase were overexpressed in M. circinelloides, up to 43% GLA was produced in the total fatty acids, and the yield of GLA reached 180 mg/l, which were, respectively, 38 and 33% higher than the control strain. These findings revealed that delta-6 desaturase (especially for delta-6-1 desaturase) plays an important role in GLA synthesis by M. circinelloides. The strain overexpressing delta-6-1 desaturase may have potential application in microbial GLA production.

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.

Endophytic fungi including black aspergilli have the potential to synthesize multiple bioactive secondary metabolites. Therefore, the search for active metabolites from endophytic fungi against pathogenic microbes has become a necessity for alternative and promising strategies. In this study, 25 endophytic fungal isolates associated with Malus domestica were isolated, grown, and fermented on a solid rice medium. Subsequently, their ethyl acetate crude extracts were pretested for biological activity. One endophytic fungal isolate demonstrated the highest activity and was chosen for further investigation. Based on its phenotypic, ITS ribosomal gene sequences, and phylogenetic characterization, this isolate was identified as Aspergillus tubingensis strain AN103 with the accession number (KR184138). Chemical investigations of its fermented cultures yielded four compounds: Pyranonigrin A (1), Fonsecin (2), TMC 256 A1 (3), and Asperazine (4). Furthermore, ¹H-NMR, HPLC, and LC-MS were performed for the identification and structure elucidation of these metabolites. The isolated pure compounds showed moderate-to-potent antibacterial activities against Pseudomonas aeruginosa and Escherichia coli (MIC value ranged from 31 and 121 to 14.5 and 58.3 μg/mL), respectively; in addition, the time–kill kinetics for the highly sensitive bacteria against isolated compounds was also investigated. The antifungal activity results show that (3) and (4) had the maximum effect against Fusarium solani and A. niger with inhibition zones of 16.40 ± 0.55 and 16.20 ± 0.20 mm, respectively, and (2) had the best effect against Candida albicans, with an inhibition zone of 17.8 ± 1.35 mm. Moreover, in a cytotoxicity assay against mouse lymphoma cell line L5178Y, (4) exhibited moderate cytotoxicity (49% inhibition), whereas (1–3) reported weak cytotoxicity (15, 26, and 19% inhibition), respectively. Our results reveal that these compounds might be useful to develop potential cytotoxic and antimicrobial drugs and an alternative source for various medical and pharmaceutical fields.

In our previous work, we reported a novel approach for increasing lipid production in an oleaginous fungus Mucor circinelloides by overexpression of mitochondrial malate transporter protein. This transporter plays a vital role in fatty acid biosynthesis during malate and citrate transport systems in oleaginous fungi. In this study, the controlling metabolic supplementation strategy was used to improve the lipid production by overexpression of malate transporter protein in M. circinelloides strain coded as Mc-MT-2. The effects of different metabolic intermediates on lipid production in batch fermentation by Mc-MT-2 were investigated. The optimal lipid production was obtained at 0.8% malic acid after 24 h of fermentation. Furthermore, in fed-batch bioreactors containing glucose as a carbon source supplemented with malic acid, the highest cell growth, and lipid production were achieved. The resulting strain showed the fungal dry biomass of 16 g/L, a lipid content of 32%, lipid yield of 5.12 g/L in a controlled bench-top bioreactor, with 1.60-, 1.60- and 2.56-fold improvement, respectively, compared with the batch control without supplementation of malic acid. Our findings revealed that the addition of malic acid during fermentation might play an important role in lipid accumulation in the recombinant M. circinelloides Mc-MT-2. This study provides valuable insights for enhanced microbial lipid production through metabolic supplementation strategy in large scale and industrial applications.