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

Abstract

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.

Spatial and temporal patterns of microphytobenthos communities along the marine-terrestrial boundary in the German Wadden Sea

“…This indicates that heterocystous cyanobacteria were more distributed in salt marsh MPB communities, an observation also commonly reported for other temperate salt marsh systems (Tracy and South, 1989;Calvo et al, 1999;Janousek, 2009). However, higher plants can also be a source of canthaxanthin (Naz et al, 2021); therefore, our results should be regarded with care despite our efforts to remove all visible plant material from sampled cores. The presence of canthaxanthin only on Spiekeroog was likely due to the fact that only the Spiekeroog transects incorporated saltmarsh sites in contrast to the other two islands comprising only tidal flat samples.…”

Biosynthesis of Naringenin from p-Coumaric Acid in Engineering Oleaginous Filamentous Fungus Mucor circinelloides

“…M. circinelloides is a typical oleaginous fungus with the potential to be as a cell factory to produce many high-value chemicals and fuels, including polyunsaturated fatty acids such as γ-linolenic acid, dihomo γ-linolenic acid, stearidonic acid, lipid, β-carotene, astaxanthin, and ethanol. − It is also agreeable for decreasing bioprocess costs, because M. circinelloides can grow on cheap substrates such as lignocellulosic biomass, gutter oil, and industrial wastewater.…”

“…To our knowledge, so far, M. circinelloides has only been used as a cell factory to produce some natural products derived from fatty acids or β-carotene including stearidonic acid, zeaxanthin, and canthaxanthin. − In these works, only one or two heterologous genes were transformed into the genome of M. circinelloides.…”

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

“…The quantitative RT-PCR was executed with the help of Light Cycler 96 Instrument (Roche Diagnostics GmbH, Basel, Switzerland) and Maxima SYBR Green qPCR Master Mix (Thermo scientific, Waltham, MA, USA) according to instructions of the manufacturer. Using the method of 2 −ΔΔCt and actin as housekeeping gene, data quantification was performed as described previously [ 34 ].…”