Screening of Structurally Distinct Lycopene β-Cyclases for Production of the Cyclic C40 Carotenoids β-Carotene and Astaxanthin by Corynebacterium glutamicum

Abstract

Lycopene β-cyclase (EC 5.5.1.19) is one of the key enzymes in the biosynthesis of β-carotene and derived carotenoids. It catalyzes isomerase reactions to form β-carotene from lycopene by β-cyclization of both of its ψ-ends. Lycopene β-cyclases are widespread in nature. We systematically analyzed the phylogeny of lycopene β-cyclases from all kingdoms of life and predicted their transmembrane structures. To this end, a collection of previously characterized lycopene β-cyclase polypeptide sequences served as bait sequences to identify their closest homologues in a range of bacteria, archaea, fungi, algae, and plant species. Furthermore, a DeepTMHMM scan was applied to search for the presence of transmembrane domains. A phylogenetic tree suggests at least five distinct clades, and the DeepTMHMM scan revealed that lycopene β-cyclases are a group of structurally different proteins: membrane-bound and cytosolic enzymes. Representative lycopene β-cyclases were screened in the lycopene-overproducing Corynebacterium glutamicum strain for β-carotene and astaxanthin production. This systematic screening facilitates the identification of new enzymes for carotenoid production. Higher astaxanthin production and less reduction of total carotenoids were achieved with the cytosolic lycopene β-cyclase CrtL from Synechococcus elongatus and the membrane-bound heterodimeric lycopene β-cyclase CrtYcd from Brevibacterium linens.

Enhancing astaxanthin biosynthesis and pathway expansion towards glycosylated C40 carotenoids by Corynebacterium glutamicum

“…In a recent study, alternative lycopene β-cyclases were identified that show a mitigated effect towards a possible feedback inhibition by various oxy-functionalized cyclic C40 carotenoids from astaxanthin biosynthesis, resulting in an improved total carotenoid biosynthesis in C. glutamicum 45 . Another study with H. pluvialis suggested that free astaxanthin could potentially inhibit its own biosynthesis and therefore the production of astaxanthin variants such as esterified astaxanthin was recommended 56 .…”

“…It has been metabolically engineered for efficient production of various non-natural C40 carotenoids including astaxanthin 12 , 44 . Recently, a screening of structurally distinct lycopene β-cyclases revealed that higher astaxanthin production can be achieved with cytosolic lycopene β-cyclase from Synechococcus elongatus and membrane-bound heterodimeric lycopene β-cyclase from Brevibacterium linens 45 . However, upon expression of β-carotene hydroxylase or β-carotene ketolase genes, a reduction of total carotenoid occurs, which might be due to a yet to be defined feedback-inhibition of the carotenoid pathway upstream of β-carotene 45 .…”

Bioflavour 2022 - Biotechnology of Flavours, Fragrances, and Functional Ingredients

“…The authors furthermore discovered a significant decrease in the total carotenoid content in the astaxanthin-producing strains and postulated a feedback inhibition of lycopene β-cyclases by astaxanthin or intermediates to be responsible for this effect. 6 The article "High-Yield Biosynthesis of trans-Nerolidol from Sugar and Glycerol" impressively demonstrates that the construction and optimization of highly efficient microbial cell factories still require a great deal of effort for testing high numbers of enzymes, strain modifications, and production parameters. The work aiming at Escherichia coli-based production of the sesquiterpene alcohol trans-nerolidol finally yielded a process that produced the pure trans form of nerolidol and could reach >90% of the theoretically possible product/substrate yield and a maximal yield of 16 g L −1 within 4 days.…”

“…A cytosolic cyclase from Synechococcus elongatus was found to be superior to the previously used enzyme from Pantoea ananatis , and a membrane-bound heterodimeric cyclase from Brevibacterium linens increased the concentrations of astaxanthin by 45%. The authors furthermore discovered a significant decrease in the total carotenoid content in the astaxanthin-producing strains and postulated a feedback inhibition of lycopene β-cyclases by astaxanthin or intermediates to be responsible for this effect …”

Astaxanthin Isomers: A Comprehensive Review of Isomerization Methods and Analytic Techniques

“…Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) is a bioactive natural pigment with red-orange color that is part of the carotenoid family, widely used in the fields of nutraceuticals, cosmetics, and animal feed. − Nowadays, quantities of astaxanthin can be industrially obtained by microorganisms. − Structurally, astaxanthin contains multiple conjugated double bonds and chiral carbon atoms, and this unique structure endows it with geometric and optical isomers (Figure ). Optical isomers of astaxanthin widely exist in various biological sources; e.g., (3 S ,3′ S ) is the predominant stereoisomer in Haematococcus pluvialis (H.…”