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In this study, 18 standard amino acids were tested as a single nitrogen source on biomass, total lipid, total fatty acid (TFA) production, and yield of γ-linolenic acid (GLA) in Rhizomucor pusillus AUMC 11616.A and Mucor circinelloides AUMC 6696.A isolated from unusual habitats. Grown for 4 days at 28°C, shaking at 150 rpm, the maximum fungal biomass for AUMC 6696.A was 14.6 ± 0.2 g/L with arginine and 13.68 ± 0.1 g/L with asparagine, when these amino acids were used as single nitrogen sources, while AUMC 11616.A maximum biomass was 10.73 ± 0.8 g/L with glycine and 9.44 ± 0.6 g/L with valine. These were significantly higher than the ammonium nitrate control ( p < 0.05). The highest levels of TFA were achieved with glycine for AUMC 11616.A, 26.2 ± 0.8% w/w of cell dry weight, and glutamic acid for AUMC 6696.A, 23.1 ± 1.3%. The highest GLA yield was seen with proline for AUMC 11616.A, 13.4 ± 0.6% w/w of TFA, and tryptophan for AUMC 6696.A, 12.8 ± 0.3%, which were 38% and 25% higher than the ammonium tartrate control. The effects of environmental factors such as temperature, pH, fermentation time, and agitation speed on biomass, total lipids, TFA, and GLA concentration of the target strains have also been investigated. Our results demonstrated that nitrogen assimilation through amino acid metabolism, as well as the use of glucose as a carbon source and abiotic factors, are integral to increasing the oleaginicity of tested strains. Few studies have addressed the role of amino acids in fermentation media, and this study sheds light on R. pusillus and M. circinelloides as promising candidates for the potential applications of amino acids as nitrogen sources in the production of lipids., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mohamed, Awad, Shah, Nazir, Naz, Hassane, Nosheen and Song.)

Thraustochytrids, such as Aurantiochytrium and Schizochytrium , have been shown as a promising sustainable alternative to fish oil due to its ability to accumulate a high level of docosahexaenoic acid (DHA) from its total fatty acids. However, the low DHA volumetric yield by most of the wild type (WT) strain of thraustochytrids which probably be caused by the low oxidative stress tolerance as well as a limited supply of key precursors for DHA biosynthesis has restricted its application for industrial application. Thus, to enhance the DHA production, we aimed to generate Aurantiochytrium SW1 mutant with high tolerance toward oxidative stress and high glucose-6 phosphate dehydrogenase (G6PDH) activities through strategic plasma mutagenesis coupled with chemical screening. The WT strain ( Aurantiochytrium sp. SW1) was initially exposed to plasma radiation and was further challenged with zeocin and polydatin, generating a mutant (YHPM1) with a 30, 65, and 80% higher overall biomass, lipid, and DHA production in comparison with the parental strains, respectively. Further analysis showed that the superior growth, lipid, and DHA biosynthesis of the YHMP1 were attributed not only to the higher G6PDH and enzymes involved in the oxidative defense such as superoxide dismutase (SOD) and catalase (CAT) but also to other key metabolic enzymes involved in lipid biosynthesis. This study provides an effective approach in developing the Aurantiochytrium sp. mutant with superior DHA production capacity that has the potential for industrial applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nazir, Phabakaran, Halim, Mohamed, Naz, Abdul Hamid and Song.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Naz, Yang, Nosheen, Sun, Nazir, Mohamed, Fazili, Ullah, Li, Yang, Garre and Song.)