Heterologous Carotenoid-Biosynthetic Enzymes: Functional Complementation and Effects on Carotenoid Profiles in Escherichia coli

Abstract

bA limited number of carotenoid pathway genes from microbial sources have been studied for analyzing the pathway complementation in the heterologous host Escherichia coli. In order to systematically investigate the functionality of carotenoid pathway enzymes in E. coli, the pathway genes of carotenogenic microorganisms (Brevibacterium linens, Corynebacterium glutamicum, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodopirellula baltica, and Pantoea ananatis) were modified to form synthetic expression modules and then were complemented with Pantoea agglomerans pathway enzymes (CrtE, CrtB, CrtI, CrtY, and CrtZ). The carotenogenic pathway enzymes in the synthetic modules showed unusual activities when complemented with E. coli. For example, the expression of heterologous CrtEs of B. linens, C. glutamicum, and R. baltica influenced P. agglomerans CrtI to convert its substrate phytoene into a rare product-3,4,3=,4=-tetradehydrolycopene-along with lycopene, which was an expected product, indicating that CrtE, the first enzyme in the carotenoid biosynthesis pathway, can influence carotenoid profiles. In addition, CrtIs of R. sphaeroides and R. capsulatus converted phytoene into an unusual lycopene as well as into neurosporene. Thus, this study shows that the functional complementation of pathway enzymes from different sources is a useful methodology for diversifying biosynthesis as nature does.

IdsA is the major geranylgeranyl pyrophosphate synthase involved in carotenogenesis in Corynebacterium glutamicum

“…By deletion of chromosomal copies, deregulation of carotenoid biosynthesis was achieved, leading to strongly elevated levels of carotenoid. Thus, the early reaction steps in a carotenoid biosynthetic pathway can influence the carotenoid yield and profile decisively . Further improvements in yield may be achieved by overexpressing and deregulating IPP‐producing operons.…”

“…Further improvements in yield may be achieved by overexpressing and deregulating IPP‐producing operons. The further use of modifying enzymes from other sources might allow the generation of novel carotenoids not found in nature when the genes encoding these enzymes are coexpressed with other carotenogenic genes in heterologous hosts .…”

Microbial Pigments From Bacteria, Yeasts, Fungi, and Microalgae for the Food and Feed Industries

“…The success of functional color expression in transgenic E. coli for the cloning of a number of carotenoid biosynthesizing genes demonstrates that enzymes from phylogenetically distant species can assemble into a functional membrane-bound multienzyme complex at which carotenoid biosynthesis takes place. Both phytoene desaturase (crtI) and lycopene cyclase (crtY) genes have been targeted for in vitro evolution to achieve synthesis of novel carotenoids in E. coli (Song et al, 2013). A variant enzyme, a desaturase chimera, efficiently catalyzed the extended desaturation of the linear C 40 carotenoid pathway, introducing six rather than four double bonds into phytoene, thus enabling the production of the fully conjugated carotenoid, 3,4,3′,4′-tetradehydrolycopene (Song et al, 2013).…”

“…Both phytoene desaturase (crtI) and lycopene cyclase (crtY) genes have been targeted for in vitro evolution to achieve synthesis of novel carotenoids in E. coli (Song et al, 2013). A variant enzyme, a desaturase chimera, efficiently catalyzed the extended desaturation of the linear C 40 carotenoid pathway, introducing six rather than four double bonds into phytoene, thus enabling the production of the fully conjugated carotenoid, 3,4,3′,4′-tetradehydrolycopene (Song et al, 2013).…”

Engineering microbial cell factories: Metabolic engineering of Corynebacterium glutamicum with a focus on non‐natural products

“…The heterologous expression of C. glutamicum carotenogenic genes has been conducted in several studies [125, 126, 130, 131], but also the potential of this Gram‐positive organism for carotenoid overproduction has been investigated [43, 128] (Fig. 1).…”