Genetic modification of Mucor circinelloides for canthaxanthin production by heterologous expression of β-carotene ketolase gene

11 pags, 4 figs, 1 tab. -- he Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnut.2021.756218/full#supplementary-materiaCanthaxanthin 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.This work was supported by the National Natural Science Foundation of China (Grant Nos. 31670064 and 31972851), the Tai Shan Industrial Experts Program tscy 20160101, and the Shandong provincial key technology R&D plan (2018GNC110039, 2018GSF121013).Peer reviewe

Role of Cytosolic Malic Enzyme in Oleaginicity of High-Lipid-Producing Fungal Strain Mucor circinelloides WJ11

Mucor circinelloides, an oleaginous filamentous fungus, is gaining popularity due to its ability to synthesize significant amounts of lipids containing γ-linolenic acid (GLA) that have important health benefits. Malic enzyme (ME), which serves as the main source of NADPH in some fungi, has been found to regulate lipid accumulation in oleaginous fungi. In the present study, the role of two cytosolic ME genes, cmalA and cmalB, in the lipid accumulation of the M. circinelloides high-lipid-producing strain WJ11, was evaluated. Strains overexpressing cmalA and cmalB showed a 9.8- and 6.4-fold rise in specific ME activity, respectively, and an elevation of the lipid content by 23.2% and 5.8%, respectively, suggesting that these genes are involved in lipid biosynthesis. Due to increased lipid accumulation, overall GLA content in biomass was observed to be elevated by 11.42% and 16.85% in cmalA and cmalB overexpressing strains, respectively. Our study gives an important insight into different studies exploring the role of the cmalA gene, while we have for the first time investigated the role of the cmalB gene in the M. circinelloides WJ11 strain

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

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