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

Tahira Naz,Samee Ullah,Hassan Mohamed,Yuanda Song,Shaoqi Li,Abu Bakr Ahmad Fazili,Caili Sun,Wu Yang,Shaista Nosheen,Victoriano Garre,Junhuan Yang,Yusuf Nazir

doi:10.3389/fnut.2021.756218

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.

Highlights

Canthaxanthin is a valuable ketocarotenoid, which is naturally present in algae, bacteria, and some fungi [1]
The strong promoter zrt1 was used for the overexpression of bkt gene
No astaxanthin could be produced due to the absence of ketolase enzymes [11]. β-carotene can be further converted into canthaxanthin by β-carotene ketolase encoded by gene bkt in algae and crtW in bacteria, which add keto group in the ring of β-carotene at 4,4-position

Summary

Introduction

Canthaxanthin is a valuable ketocarotenoid, which is naturally present in algae, bacteria, and some fungi [1]. Canthaxanthin is commonly present in wild bird tissue, egg yolk, crustaceans, and fish at low levels. It was first isolated as a major coloring pigment from the edible mushroom Cantharellus cinnabarinus, from where it was named canthaxanthin [2]. It is formed as an intermediate compound in β-carotene metabolisms to astaxanthin. It has more effective antioxidant potential than βcarotene [3] due to which it is regarded as one among the important xanthophylls of commercial significance and has extensive utilization in industries [4]. It is used as a feed additive to obtain red color in egg yolks and skins [1], while in the cosmetics industry, it is used as a pigmenting agent for human skin applications

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Results

Conclusion