High-Level Production of the Industrial Product Lycopene by the Photosynthetic Bacterium Rhodospirillum rubrum

Abstract

bThe biosynthesis of the major carotenoid spirilloxanthin by the purple nonsulfur bacterium Rhodospirillum rubrum is thought to occur via a linear pathway proceeding through phytoene and, later, lycopene as intermediates. This assumption is based solely on early chemical evidence (B. H. Davies, Biochem. J. 116:93-99, 1970). In most purple bacteria, the desaturation of phytoene, catalyzed by the enzyme phytoene desaturase (CrtI), leads to neurosporene, involving only three dehydrogenation steps and not four as in the case of lycopene. We show here that the chromosomal insertion of a kanamycin resistance cassette into the crtCcrtD region of the partial carotenoid gene cluster, whose gene products are responsible for the downstream processing of lycopene, leads to the accumulation of the latter as the major carotenoid. We provide spectroscopic and biochemical evidence that in vivo, lycopene is incorporated into the light-harvesting complex 1 as efficiently as the methoxylated carotenoids spirilloxanthin (in the wild type) and 3,4,3=,4=-tetrahydrospirilloxanthin (in a crtD mutant), both under semiaerobic, chemoheterotrophic, and photosynthetic, anaerobic conditions. Quantitative growth experiments conducted in dark, semiaerobic conditions, using a growth medium for high cell density and high intracellular membrane levels, which are suitable for the conventional industrial production in the absence of light, yielded lycopene at up to 2 mg/g (dry weight) of cells or up to 15 mg/liter of culture. These values are comparable to those of many previously described Escherichia coli strains engineered for lycopene production. This study provides the first genetic proof that the R. rubrum CrtI produces lycopene exclusively as an end product.

Random mutagenesis and overexpression of rhodopin-3,4-desaturase allows the production of highly conjugated carotenoids in Rhodospirillum rubrum

“…Construction steps for the crtD expression plasmid, pRKCAG53. Digestion of the plasmid pBsSGE5 [22], which contains several carotenoid biosynthesis genes from the R. rubrum chromosome, with PstI, yielded the plasmid pBsCAG53, containing only crtD and crtE as intact genes. The insert of pBsCAG53 was then cloned into the broad-host range plasmid pRK404 to create the crtD expression plasmid pRKCAG53 (see Materials and methods).…”

“…First, the plasmid pBsSGE5 [22], which contains a 6.5 kb EcoRV fragment of R. rubrum chromosomal DNA (Fig. 2), ligated into the SmaI Fig.…”

“…We have reported previously [22] that the R. rubrum crtD À mutant ST4 can be complemented by a DNA fragment of the R. rubrum chromosome containing several genes of carotenoid biosynthesis (Fig. 2).…”

“…The production of carotenoids in purple bacteria is an interesting novel strategy for the biotechnological realisation of this class of substances [8,22]. Purple bacteria naturally produce carotenoids in high amounts and incorporate them into their photosynthetic apparatus, which consists of a light-harvesting complex (LH1) 1 and a reaction centre [19].…”

An overview on biofuel and biochemical production by photosynthetic microorganisms with understanding of the metabolism and by metabolic engineering together with efficient cultivation and downstream processing

“…PCC6803 with the aid of a β-caryophyllene synthase gene from Artemisia annua [161], where this compound is used in the fragrance and cosmetic industry, and in natural remedies for its anti-inflammatory and antimicrobial properties. Lycopene is also important food additives and pigment, and can be produced by a purple non-sulfur bacterium, R. rubrum, by deletion of downstream phytoene desaturase gene crtC and crtD [162].…”