The biotechnological potential and design of novel carotenoids by gene combination in Escherichia coli

Sandmann, Gerhard; Albrecht, Manuela; Schnurr, Georg; Knörzer, Oliver C.; Böger, Peter

doi:10.1016/s0167-7799(99)01307-4

Cited by 99 publications

(42 citation statements)

References 37 publications

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“…[24,[34][35][36][37][38]]. The bacterial system could potentially provide a simple platform to manipulate and study the CYP97 carotene hydroxylases.…”

Section: Introductionmentioning

confidence: 99%

Escherichia coli as a platform for functional expression of plant P450 carotene hydroxylases

Quinlan

Jaradat

Wurtzel

2007

Archives of Biochemistry and Biophysics

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Carotenoids and their derivatives are essential for growth, development, and signaling in plants and have an added benefit as nutraceuticals in food crops. Despite the importance of the biosynthetic pathway, there remain open questions regarding some of the later enzymes in the pathway. The CYP97 family of P450 enzymes was predicted to function in carotene ring hydroxylation, to convert provitamin A carotenes to nonprovitamin A xanthophylls. However, substrate specificity was difficult to investigate directly in plants, which mask enzyme activities by a complex and dynamic metabolic network. To characterize the enzymes more directly, we amplified cDNAs from a model crop, Oryza sativa, and used functional complementation in Escherichia coli to test activity and specificity of members of Clans A and C. This heterologous system will be valuable for further study of enzyme interactions and substrate utilization needed to understand better the role of CYP97 hydroxylases in plant carotenoid biosynthesis.

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“…[24,[34][35][36][37][38]]. The bacterial system could potentially provide a simple platform to manipulate and study the CYP97 carotene hydroxylases.…”

Section: Introductionmentioning

confidence: 99%

Escherichia coli as a platform for functional expression of plant P450 carotene hydroxylases

Quinlan

Jaradat

Wurtzel

2007

Archives of Biochemistry and Biophysics

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“…This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoidbased pathways, could lead to the synthesis of novel carotenoids and their derivatives.Carotenoids are naturally occurring pigments found in a wide variety of plants and microorganisms (22,23). Recent studies indicate that these isoprenoid-based natural products possess biologically active properties (21, 24), thereby making them of interest to the medicinal chemist.…”

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confidence: 99%

“…Carotenoids are naturally occurring pigments found in a wide variety of plants and microorganisms (22,23). Recent studies indicate that these isoprenoid-based natural products possess biologically active properties (21, 24), thereby making them of interest to the medicinal chemist.…”

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confidence: 99%

Preparation, Characterization, and Optimization of an In Vitro C₃₀Carotenoid Pathway

Jeong

Mijts

et al. 2005

Appl Environ Microbiol

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The ispA gene encoding farnesyl pyrophosphate (FPP) synthase from Escherichia coli and the crtM gene encoding 4,4-diapophytoene (DAP) synthase from Staphylococcus aureus were overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoidbased pathways, could lead to the synthesis of novel carotenoids and their derivatives.Carotenoids are naturally occurring pigments found in a wide variety of plants and microorganisms (22,23). Recent studies indicate that these isoprenoid-based natural products possess biologically active properties (21, 24), thereby making them of interest to the medicinal chemist. Most carotenoids belong to the C 30 and C 40 classes, being distinguished by the number of iterative steps of isopentenyl pyrophosphate (IPP) condensation to add C 5 isoprene units. The initial synthetic step is the condensation of IPP with ␥,␥-dimethylallyl pyrophosphate (DMAPP) catalyzed by farnesyl pyrophosphate (FPP) synthase (ispA) to give geranyl pyrophosphate (GPP) and, sequentially, FPP (9). This C 15 compound serves as a central node for the synthesis of sterols (29), farnesylated proteins (4), hemes (20), sesquiterpenes (6), and dolichols (5), as well as the crucial precursor for carotenoids.C 30 carotenoids are present in the nonphotosynthetic bacteria, such as Streptococcus faecium, Staphylococcus aureus, and Methylobacterium rhodinum, and in the photosynthetic Heliobacterium species (16,25,27,28). Only the genes encoding 4,4Ј-diapophytoene (DAP) synthase (crtM) and 4,4Ј-diapophytoene desaturase (crtN) from S. aureus have been cloned and functionally expressed in Escherichia coli, resulting in the yellow 4,4Ј-diaponeurosporene from FPP (30) (Fig. 1). The first committed step in C 30 carotenoid biosynthesis is the condensation of two molecules of FPP catalyzed by DAP synthase to form the colorless carotenoid 4,4Ј-diapophytoene.Until now, only a few reports have discussed the formation of DAP through a reconstituted C 30 carotenoid metabolic pathway in vitro (13, 17), and none have provided a full kinetic analysis or reaction optimization of the in vitro pathway. Nevertheless, such a study would be valuable in order to design novel carotenoids and carotenoid-based hybrid natural products. Therefore, in the current work, we set out to optimize the C 30 carotenoid pathway reconstructed in vitro for the synthesis of DAP from IPP and DMAPP. Furthermore, we elucidated the kinetic behavior of both the FPP synthase a...

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“…The potential commercial interest for the production of carotenoids and the cloning of genes encoding biosynthetic enzymes has led to all kinds of examples of metabolic pathway engineering. These examples include the overexpression of a gene encoding a rate-limiting enzyme (14,17), the expression of carotenogenic genes in noncarotenogenic heterologous hosts (12,18,20,32), the increase of the carbon flux into the carotenoid biosynthetic pathway (1,12,17,32), and the combination of genes and modification of catalytic activities in order to improve and/or modify carotenoid biosynthetic pathways (18,(24)(25)(26)32).…”

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Metabolic Engineering of the Carotenoid Biosynthetic Pathway in the Yeast Xanthophyllomyces dendrorhous ( Phaffia rhodozyma )

Verdoes

Sandmann

Visser

et al. 2003

Appl Environ Microbiol

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The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a ␤-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the ␤-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono-and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via ␤-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3-4-didehydro-␤--caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.

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The biotechnological potential and design of novel carotenoids by gene combination in Escherichia coli

Cited by 99 publications

References 37 publications

Escherichia coli as a platform for functional expression of plant P450 carotene hydroxylases

Escherichia coli as a platform for functional expression of plant P450 carotene hydroxylases

Preparation, Characterization, and Optimization of an In Vitro C₃₀Carotenoid Pathway

Metabolic Engineering of the Carotenoid Biosynthetic Pathway in the Yeast Xanthophyllomyces dendrorhous ( Phaffia rhodozyma )

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The biotechnological potential and design of novel carotenoids by gene combination in Escherichia coli

Cited by 99 publications

References 37 publications

Escherichia coli as a platform for functional expression of plant P450 carotene hydroxylases

Escherichia coli as a platform for functional expression of plant P450 carotene hydroxylases

Preparation, Characterization, and Optimization of an In Vitro C30Carotenoid Pathway

Metabolic Engineering of the Carotenoid Biosynthetic Pathway in the Yeast Xanthophyllomyces dendrorhous ( Phaffia rhodozyma )

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Preparation, Characterization, and Optimization of an In Vitro C₃₀Carotenoid Pathway