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Biodegradation of poly (vinyl alcohol) by an orychophragmus rhizosphere-associated fungus Penicillium brevicompactum OVR-5, and its proposed PVA biodegradation pathway

Mohamed H, Shah AM, Nazir Y, Naz T, Nosheen S, Song Y

World J Microbiol Biotechnol, 2021 Dec 06;38(1):10.
PMID: 34866162 DOI: 10.1007/s11274-021-03197-x

In recent years, the utilisation of endophytes has emerged as a promising biological treatment technology for the degradation of plastic wastes such as biodegradation of synthetic plastics. This study, therefore, aimed to explore and extensively screen endophytic fungi (from selected plants) for efficient in vitro polyvinyl alcohol (PVA) biodegradation. In total, 76 endophytic fungi were isolated and cultivated on a PVA screening agar medium. Among these fungi, 10 isolates showed potential and were subsequently identified based on phenotypical characteristics, ITS ribosomal gene sequences, and phylogenetic analyses. Four strains exhibited a maximum level of PVA-degradation in the liquid medium when cultivated for 10 days at 28 °C and 150 rpm. These strains showed varied PVA removal rates of 81% (Penicillium brevicompactum OVR-5), 67% (Talaromyces verruculosus PRL-2), 52% (P. polonicum BJL-9), and 41% (Aspergillus tubingensis BJR-6) respectively. The most promising PVA biodegradation isolate 'OVR-5', with an optimal pH at 7.0 and optimal temperature at 30 °C, produced lipase, manganese peroxidase, and laccase enzymes. Based on analyses of its metabolic intermediates, as identified with GC-MS, we proposed the potential PVA degradation pathway of OVR-5. Biodegradation results were confirmed through scanning electron microscopy and Fourier transform infrared spectroscopy. This study provides the first report on an endophytic P. brevicompactum strain (associated with Orychophragmus violaceus) that has a great ability for PVA degradation providing more insight on potential fungus-based applications in plastic waste degradation.
Fulltext Coculturing of Mucor plumbeus and Bacillus subtilis bacterium as an efficient fermentation strategy to enhance fungal lipid and gamma-linolenic acid (GLA) production

Mohamed H, Awad MF, Shah AM, Sadaqat B, Nazir Y, Naz T, et al.

Sci Rep, 2022 Jul 30;12(1):13111.
PMID: 35908106 DOI: 10.1038/s41598-022-17442-2

This study aimed to improve lipid and gamma-linolenic acid (GLA) production of an oleaginous fungus, Mucor plumbeus, through coculturing with Bacillus subtilis bacteria, optimising the environmental and nutritional culture conditions, and scaling them for batch fermentation. The maximum levels of biomass, lipid, fatty acid, and GLA in a 5 L bioreactor containing cellobiose and ammonium sulfate as the optimal carbon and nitrogen sources, respectively, achieved during the coculturing processes were 14.5 ± 0.4 g/L, 41.5 ± 1.3, 24 ± 0.8, and 20 ± 0.5%, respectively. This strategy uses cellobiose in place of glucose, decreasing production costs. The nutritional and abiotic factor results suggest that the highest production efficiency is achieved at 6.5 pH, 30 °C temperature, 10% (v/v) inoculum composition, 200 rpm agitation speed, and a 5-day incubation period. Interestingly, the GLA concentration of cocultures (20.0 ± 0.5%) was twofold higher than that of monocultures (8.27 ± 0.11%). More importantly, the GC chromatograms of cocultures indicated the presence of one additional peak corresponding to decanoic acid (5.32 ± 0.20%) that is absent in monocultures, indicating activation of silent gene clusters via cocultivation with bacteria. This study is the first to show that coculturing of Mucor plumbeus with Bacillus subtilis is a promising strategy with industrialisation potential for the production of GLA-rich microbial lipids and prospective biosynthesis of new products.
Fulltext Role of Snf-β in lipid accumulation in the high lipid-producing fungus Mucor circinelloides WJ11

Nosheen S, Naz T, Yang J, Hussain SA, Fazili ABA, Nazir Y, et al.

Microb Cell Fact, 2021 Feb 27;20(1):52.
PMID: 33639948 DOI: 10.1186/s12934-021-01545-y

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.
Fulltext Redirecting Metabolic Flux towards the Mevalonate Pathway for Enhanced β-Carotene Production in M. circinelloides CBS 277.49

Naz T, Nazir Y, Nosheen S, Ullah S, Halim H, Fazili ABA, et al.

Biomed Res Int, 2020;2020:8890269.
PMID: 33457420 DOI: 10.1155/2020/8890269

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 μg/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.
Fulltext Genetic Modification of Mucor circinelloides for Canthaxanthin Production by Heterologous Expression of β-carotene Ketolase Gene

Naz T, Yang J, Nosheen S, Sun C, Nazir Y, Mohamed H, et al.

Front Nutr, 2021;8:756218.
PMID: 34722614 DOI: 10.3389/fnut.2021.756218

Canthaxanthin is a reddish-orange xanthophyll with strong antioxidant activity and higher bioavailability than carotenes, primarily used in food, cosmetics, aquaculture, and pharmaceutical industries. The spiking market for natural canthaxanthin promoted researchers toward genetic engineering of heterologous hosts for canthaxanthin production. Mucor circinelloides is a dimorphic fungus that produces β-carotene as the major carotenoid and is considered as a model organism for carotenogenic studies. In this study, canthaxanthin-producing M. circinelloides strain was developed by integrating the codon-optimized β-carotene ketolase gene (bkt) of the Haematococcus pluvialis into the genome of the fungus under the control of strong promoter zrt1. First, a basic plasmid was constructed to disrupt crgA gene, a negative regulator of carotene biosynthesis resulted in substantial β-carotene production, which served as the building block for canthaxanthin by further enzymatic reaction of the ketolase enzyme. The genetically engineered strain produced a significant amount (576 ± 28 μg/g) of canthaxanthin, which is the highest amount reported in Mucor to date. Moreover, the cell dry weight of the recombinant strain was also determined, producing up to more than 9.0 g/L, after 96 h. The mRNA expression level of bkt in the overexpressing strain was analyzed by RT-qPCR, which increased by 5.3-, 4.1-, and 3-folds at 24, 48, and 72 h, respectively, compared with the control strain. The canthaxanthin-producing M. circinelloides strain obtained in this study provided a basis for further improving the biotechnological production of canthaxanthin and suggested a useful approach for the construction of more valuable carotenoids, such as astaxanthin.