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Astaxanthin is a bioactive natural pigment with antioxidant properties. It has extensive applications within the industrial sector as well as in human and animal health. Mucor circinelloides is a zygomycete fungus that accumulates β-carotene as the main carotenoid compound. M. circinelloides is a well-known model organism among Mucorales for studying carotenogenesis in fungi, which makes it a promising candidate for the biotechnological production of carotenoids. In this study, β-carotene hydroxylase (crtR-B) and ketolase (bkt) genes (codon-optimized) were coexpressed from Haematococcus pluvialis in M. circinelloides using two potent promoters gpd1 and zrt1 respectively to generate an astaxanthin-producing biofactory. Following 72 h of cultivation, the recombinant M. circinelloides Mc-57 obtained in this study produced 135 ± 8 µg/g of astaxanthin. This is the highest reported amount in M. circinelloides to date. The mRNA levels of crtR-B and bkt in Mc-57 were assayed using RT-qPCR. These levels showed a 5.7-fold increase at 72 h and a 5.5-fold increase at 24 h, respectively, compared to the control strain. This demonstrated the successful overexpression of both genes, which correlated with the production of astaxanthin in the Mc-57. Moreover, the addition of glutamate (2 g/L) and mevalonate (15 mM) resulted in an increase in astaxanthin production in the recombinant strain. The results showed that the combined addition of these metabolic precursors resulted in 281 ± 20 µg/g of astaxanthin, which is 2.08-fold higher than the control medium (135 ± 8 µg/g). The addition of metabolic precursors also positively impacted the biomass growth of Mc-57, reaching 11.2 ± 0.57 g/L compared to 9.1 ± 0.23 g/L (control medium). The study successfully addressed the challenge of balancing the accumulation of astaxanthin with biomass growth, which has been regarded as common bottleneck in the metabolic engineering of microbial cells. The development of a recombinant fungal strain of M. circinelloides not only increased astaxanthin content. Additionally, it provided a foundation for further improvement of the biotechnological production of astaxanthin in M. circinelloides., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

This study aimed to improve the lipid and biomass yields of Mucor circinelloides WJ11 by implementing four different fed-batch fermentation strategies, varied in time and glucose concentration (S1-S4). The S1 fermentation strategy yielded the highest biomass, lipid, and fatty acid content (22 ± 0.7 g/L, 53 ± 1.2 %, and 28 ± 1.6 %) after 120 and 144 h, respectively. The γ-linolenic acid titer of 0.75 ± 0.0 g/L was greatest in S3 after 48 h. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to analyze the transcription of key genes involved in lipid accumulation. The glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and ATP-citrate lyase genes showed increased expression levels. Fourier-transform infrared (FTIR) spectroscopy was used to analyze the biochemical profile during fermentation strategies. Optimal abiotic factors for production efficiency included pH 6.5, 25-26 °C, 15 % (v/v) inoculum, 500 rpm, 20 %-30 % dissolved oxygen, and 120 h fermentation. Glucose co-feeding offers valuable insights to develop effective fermentation strategies for lipid production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Microbial oils have gained massive attention because of their significant role in industrial applications. Currently plants and animals are the chief sources of medically and nutritionally important fatty acids. However, the ever-increasing global demand for polyunsaturated fatty acids (PUFAs) cannot be met by the existing sources. Therefore microbes, especially fungi, represent an important alternative source of microbial oils being investigated. Mucor circinelloides-an oleaginous filamentous fungus, came to the forefront because of its high efficiency in synthesizing and accumulating lipids, like γ-linolenic acid (GLA) in high quantity. Recently, mycelium of M. circinelloides has acquired substantial attraction towards it as it has been suggested as a convenient raw material source for the generation of biodiesel via lipid transformation. Although M. circinelloides accumulates lipids naturally, metabolic engineering is found to be important for substantial increase in their yields. Both modifications of existing pathways and re-formation of biosynthetic pathways in M. circinelloides have shown the potential to improve lipid levels. In this review, recent advances in various important metabolic aspects of M. circinelloides have been discussed. Furthermore, the potential applications of M. circinelloides in the fields of antioxidants, nutraceuticals, bioremediation, ethanol production, and carotenoids like beta carotene and astaxanthin having significant nutritional value are also deliberated., (© 2022. The Author(s).)

Background: Mucor circinelloides WJ11 is a high-lipid producing strain and an excellent producer of γ-linolenic acid (GLA) which is crucial for human health. We have previously identified genes that encode for AMP-activated protein kinase (AMPK) complex in M. circinelloides which is an important regulator for lipid accumulation. Comparative transcriptional analysis between the high and low lipid-producing strains of M. circinelloides showed a direct correlation in the transcriptional level of AMPK genes with lipid metabolism. Thus, the role of Snf-β, which encodes for β subunit of AMPK complex, in lipid accumulation of the WJ11 strain was evaluated in the present study., Results: The results showed that lipid content of cell dry weight in Snf-β knockout strain was increased by 32 % (from 19 to 25 %). However, in Snf-β overexpressing strain, lipid content of cell dry weight was decreased about 25 % (from 19 to 14.2 %) compared to the control strain. Total fatty acid analysis revealed that the expression of the Snf-β gene did not significantly affect the fatty acid composition of the strains. However, GLA content in biomass was increased from 2.5 % in control strain to 3.3 % in Snf-β knockout strain due to increased lipid accumulation and decreased to 1.83 % in Snf-β overexpressing strain. AMPK is known to inactivate acetyl-CoA carboxylase (ACC) which catalyzes the rate-limiting step in lipid synthesis. Snf-β manipulation also altered the expression level of the ACC1 gene which may indicate that Snf-β control lipid metabolism by regulating ACC1 gene., Conclusions: Our results suggested that Snf-β gene plays an important role in regulating lipid accumulation in M. circinelloides WJ11. Moreover, it will be interesting to evaluate the potential of other key subunits of AMPK related to lipid metabolism. Better insight can show us the way to manipulate these subunits effectively for upscaling the lipid production. Up to our knowledge, it is the first study to investigate the role of Snf-β in lipid accumulation in M. circinelloides.

Background: AMP-activated protein kinase (AMPK) is an important regulator for lipid accumulation, potentially known to have an inhibitory role in lipid synthesis. It inactivates acetyl-CoA carboxylase (ACC), an important regulatory enzyme required for lipid synthesis. However, in Mucor circinelloides, AMPK and its association with lipid accumulation has not been studied yet., Objectives: To identify AMPK genes in M. circinelloides and to compare their expression levels in high and low lipid-producing strains of M. circinelloides to predict the possible roles of AMPK in lipid metabolism and to select candidate genes for further studies to enhance lipid accumulation., Results: Two genes for α-subunit, one for β-subunit and six for γ-subunit were identified and annotated. Bioinformatic analysis confirmed the presence of typical conserved domains in these genes. Furthermore, transcriptional profiling displayed marked differences in expression kinetics of subunits among the selected strains. The expression of AMPK genes decreased rapidly in WJ11, high lipid producer strain during the lipid accumulation phase while contrasting profile of expression was observed in CBS 277.49, low lipid producer strain., Conclusion: The present study has shown the association of AMPK genes with lipid metabolism at the transcriptional level. The involvement of Snf-α1, Snf-α2, Snf-β, Snf-γ1, Snf-γ4, Snf-γ5 subunits were shown to be more pronounced and could potentially be further explored in future studies.

Carotenoids produced by microbial sources are of industrial and medicinal importance due to their antioxidant and anticancer properties. In the current study, optimization of β -carotene production in M. circinelloides strain 277.49 was achieved using response surface methodology (RSM). Cerulenin and ketoconazole were used to inhibit fatty acids and the sterol biosynthesis pathway, respectively, in order to enhance β -carotene production by diverting metabolic pool towards the mevalonate pathway. All three variables used in screening experiments were found to be significant for the production of β -carotene. The synergistic effect of the C/N ratio, cerulenin, and ketoconazole was further evaluated and optimized for superior β -carotene production using central composite design of RSM. Our results found that the synergistic combination of C/N ratios, cerulenin, and ketoconazole at different concentrations affected the β -carotene productions significantly. The optimal production medium (std. order 11) composed of C/N 25, 10  μ g/mL cerulenin, and 150 mg/L ketoconazole, producing maximum β -carotene of 4.26 mg/L (0.43 mg/g) which was 157% greater in comparison to unoptimized medium (1.68 mg/L, 0.17 mg/g). So, it was concluded that metabolic flux had been successfully redirected towards the mevalonate pathway for enhanced β -carotene production in CBS 277.49., Competing Interests: The authors declare no conflicting interests., (Copyright © 2020 Tahira Naz et al.)

γ -Linolenic acid (GLA) and carotenoids have attracted much interest due to their nutraceutical and pharmaceutical importance. Mucoromycota, typical oleaginous filamentous fungi, are known for their production of valuable essential fatty acids and carotenoids. In the present study, 81 fungal strains were isolated from different Egyptian localities, out of which 11 Mucoromycota were selected for further GLA and carotenoid investigation. Comparative analysis of total lipids by GC of selected isolates showed that GLA content was the highest in Rhizomucor pusillus AUMC 11616.A, Mucor circinelloides AUMC 6696.A, and M. hiemalis AUMC 6031 that represented 0.213, 0.211, and 0.20% of CDW, respectively. Carotenoid analysis of selected isolates by spectrophotometer demonstrated that the highest yield of total carotenoids (640  μ g/g) was exhibited by M. hiemalis AUMC 6031 and M. hiemalis AUMC 6695, and these isolates were found to have a similar carotenoid profile with, β -carotene (65%), zeaxanthin (34%), astaxanthin, and canthaxanthin (5%) of total carotenoids. The total fatty acids of all tested isolates showed moderate antimicrobial activity against Staphylococcus aureus and Salmonella Typhi , and Penicillium chrysogenum . To the best of our knowledge, this is the first report on the highest yield of total lipid accumulation (51.74% CDW) by a new oleaginous fungal isolate R. pusillus AUMC 11616.A. A new scope for a further study on this strain will be established to optimize and improve its total lipids with high GLA production. So, R. pusillus AUMC 11616.A might be a potential candidate for industrial application., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Hassan Mohamed et al.)