decaprenoxanthin and astaxanthine

Corynebacterium glutamicum is a workhorse of industrial amino acid production employed for more than five decades for the million-ton-scale production of L-glutamate and L-lysine. This bacterium is pigmented due to the biosynthesis of the carotenoid decaprenoxanthin. Decaprenoxanthin is a carotenoid with 50 carbon atoms, and, thus, C. glutamicum belongs to the rare group of bacteria that produce long-chain C50 carotenoids. C50 carotenoids have been mainly isolated from extremely halophilic archaea (Kelly and Jensen, Acta Chem Scand 21:2578, 1967; Pfander, Pure Appl Chem 66:2369-2374, 1994) and from Gram-positive bacteria of the order Actinomycetales (Netzer et al., J Bacteriol 192:5688-5699, 2010). The characteristic yellow phenotype of C. glutamicum is due to the cyclic C50 carotenoid decaprenoxanthin and its glycosides. Decaprenoxanthin production has been improved by plasmid-borne overexpression of endogenous genes of carotenogenesis. Gene deletion resulted in the production of the C40 carotenoid lycopene, an intermediate of decaprenoxanthin biosynthesis. Heterologous gene expression was required to develop strains overproducing nonnative carotenoids and terpenes, such as astaxanthin (Henke et al., Mar Drugs 14:E124, 2016) and (+)-valencene (Frohwitter et al., J Biotechnol 191:205-213, 2014). Integration of additional copies of endogenous genes expressed from strong promoters improved isoprenoid biosynthesis. Here, we describe C. glutamicum strains, plasmids, and methods for overexpression of endogenous and heterologous genes, gene deletion, replacement, and genomic integration. Moreover, strain cultivation as well as extraction, identification, and quantitative determination of terpenes and carotenoids produced by C. glutamicum is detailed.

Topics: Carotenoids; Chromatography, High Pressure Liquid; Corynebacterium glutamicum; Gas Chromatography-Mass Spectrometry; Gene Deletion; Genetic Engineering; Genomics; Liquid-Liquid Extraction; Metabolic Engineering; Terpenes; Transformation, Genetic; Xanthophylls

Astaxanthin, a red C40 carotenoid, is one of the most abundant marine carotenoids. It is currently used as a food and feed additive in a hundred-ton scale and is furthermore an attractive component for pharmaceutical and cosmetic applications with antioxidant activities. Corynebacterium glutamicum, which naturally synthesizes the yellow C50 carotenoid decaprenoxanthin, is an industrially relevant microorganism used in the million-ton amino acid production. In this work, engineering of a genome-reduced C. glutamicum with optimized precursor supply for astaxanthin production is described. This involved expression of heterologous genes encoding for lycopene cyclase CrtY, β-carotene ketolase CrtW, and hydroxylase CrtZ. For balanced expression of crtW and crtZ their translation initiation rates were varied in a systematic approach using different ribosome binding sites, spacing, and translational start codons. Furthermore, β-carotene ketolases and hydroxylases from different marine bacteria were tested with regard to efficient astaxanthin production in C. glutamicum. In shaking flasks, the C. glutamicum strains developed here overproduced astaxanthin with volumetric productivities up to 0.4 mg·L(-1)·h(-1) which are competitive with current algae-based production. Since C. glutamicum can grow to high cell densities of up to 100 g cell dry weight (CDW)·L(-1), the recombinant strains developed here are a starting point for astaxanthin production by C. glutamicum.

Topics: beta Carotene; Carotenoids; Corynebacterium glutamicum; Metabolic Engineering; Mixed Function Oxygenases; Oxygenases; Xanthophylls