Simple functional analysis of key genes involved in astaxanthin biosynthesis using Arabidopsis cultured cells

Harada, Hisashi; Fujisawa, Masaki; Teramoto, Maki; Sakurai, Nozomu; Suzuki, Hideyuki; Shibata, Daisuke; Misawa, Norihiko

doi:10.5511/plantbiotechnology.26.81

Cited by 7 publications

(8 citation statements)

References 63 publications

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“…Isolation of the genes that encode pathway enzymes, and modifying their expression, constitutes one approach to overcome this challenge, but modulating single enzymes is often unhelpful because the pathways are regulated at multiple points. It has been shown that the simultaneous expression of multiple enzymes is the most desirable way to study and modulate complex pathways such as carotenoid biosynthesis (Zhu et al 2008;Harada et al 2009;Fujisawa et al 2009;Naqvi et al 2009). In this study, Lilium 9 formolongi was successfully transformed by the introduction of seven bacterial enzyme genes.…”

Section: Discussionmentioning

confidence: 99%

“…strain SD212 (crtZ and crtW) and Paracoccus sp. strain N81106 (formerly called Agrobacterium aurantiacum; idi) Harada et al 2009). For inoculation, A. tumefaciens strain EHA101 containing the plasmid was used.…”

Section: Bacterial Strain and Plasmid Constructmentioning

confidence: 99%

“…The carotenoid biosynthetic pathway in crops has been modified to increase carotenoid levels by genetic manipulation using key genes in carotenoid biosynthesis such as the phytoene synthase gene (crtB or Psy) (Shewmaker et al 1999;Ye et al 2000;Rosati et al 2000;Fraser et al 2002;Ducreux et al 2005;Paine et al 2005;Diretto et al 2006Diretto et al , 2007aFujisawa et al 2008;Naqvi et al 2009;Apel and Bock 2009). Recently, pathway engineering of plants for producing novel ketocarotenoids, that is carotenoids with 4-ketolated b-ring, has been the focus of many researchers of plant biotechnology, since ketocarotenoids including astaxanthin and canthaxanthin confer special benefits on human health (Mann et al 2000;Stalberg et al 2003;Ralley et al 2004;Morris et al 2006;Gerjets et al 2007;Zhu et al 2008;Hasunuma et al 2008;Harada et al 2009;Fujisawa et al 2009). Flower color is also changed from yellow to red by synthesizing ketocarotenoids in the petal (Suzuki et al 2007).…”

Section: Introductionmentioning

confidence: 99%

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Metabolic engineering of Lilium × formolongi using multiple genes of the carotenoid biosynthesis pathway

Azadi

Otang

Chin

et al. 2010

Plant Biotechnol Rep

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Lilium 9 formolongi was genetically engineered by Agrobacterium-mediated transformation with the plasmid pCrtZW-N8idi-crtEBIY, which contains seven enzyme genes under the regulation of the CaMV 35S promoter. In the transformants, ketocarotenoids were detected in both calli and leaves, which showed a strong orange color. In transgenic calli, the total amount of carotenoids [133.3 lg/g fresh weight (FW)] was 26.1-fold higher than in wild-type calli. The chlorophyll content and photosynthetic efficiency in transgenic orange plantlets were significantly lowered; however, after several months of subculture, they had turned into plantlets with green leaves that showed significant increases in chlorophyll and photosynthetic efficiency. The total carotenoid contents in leaves of transgenic orange and green plantlets were quantified at 102.9 and 135.2 lg/g FW, respectively, corresponding to 5.6-and 7.4-fold increases over the levels in the wild-type. Ketocarotenoids such as echinenone, canthaxanthin, 3 0 -hydroxyechinenone, 3-hydroxyechinenone, and astaxanthin were detected in both transgenic calli and orange leaves. A significant change in the type and composition of ketocarotenoids was observed during the transition from orange transgenic plantlets to green plantlets. Although 3 0 -hydroxyechinenone, 3-hydroxyechinenone, astaxanthin, and adonirubin were absent, and echinenone and canthaxanthin were present at lower levels, interestingly, the upregulation of carotenoid biosynthesis led to an increase in the total carotenoid concentration (?31.4%) in leaves of the transgenic green plantlets.

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Section: Discussionmentioning

confidence: 99%

Section: Bacterial Strain and Plasmid Constructmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Metabolic engineering of Lilium × formolongi using multiple genes of the carotenoid biosynthesis pathway

Azadi

Otang

Chin

et al. 2010

Plant Biotechnol Rep

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“…SD212 have been chosen (Choi et al , ). This approach has been adopted on the basis of previous reports, which demonstrated the efficacy of Brevundimonas CrtW and CrtZ in enabling transgenic higher plants to produce large quantities of Asx (Hasunuma et al , Harada et al , , Mortimer et al , Nogueira et al ). By contrast, previous attempts at engineering ketocarotenoid biosynthesis in cyanobacteria by introducing carotenogenic enzymes from different organisms (Harker and Hirschberg ) and/or additional copies of the endogenous ones (Albers ) have not been as successful.…”

Section: Discussionmentioning

confidence: 99%

Non‐endogenous ketocarotenoid accumulation in engineeredSynechocystissp. PCC 6803

Menin

Santabarbara

Lami³

et al. 2019

Physiologia Plantarum

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The cyanobacterium Synechocystis sp. PCC 6803 is a model species commonly employed for biotechnological applications. It is naturally able to accumulate zeaxanthin (Zea) and echinenone (Ech), but not astaxanthin (Asx), which is the highest value carotenoid produced by microalgae, with a wide range of applications in pharmaceutical, cosmetics, food and feed industries. With the aim of finding an alternative and sustainable biological source for the production of Asx and other valuable hydroxylated and ketolated intermediates, the carotenoid biosynthetic pathway of Synechocystis sp. PCC 6803 has been engineered by introducing the 4,4′ β‐carotene oxygenase (CrtW) and 3,3′ β‐carotene hydroxylase (CrtZ) genes from Brevundimonas sp. SD‐212 under the control of a temperature‐inducible promoter. The expression of exogenous CrtZ led to an increased accumulation of Zea at the expense of Ech, while the expression of exogenous CrtW promoted the production of non‐endogenous canthaxanthin and an increase in the Ech content with a concomitant strong reduction of β‐carotene (β‐car). When both Brevundimonas sp. SD‐212 genes were coexpressed, significant amounts of non‐endogenous Asx were obtained accompanied by a strong decrease in β‐car content. Asx accumulation was higher (approximately 50% of total carotenoids) when CrtZ was cloned upstream of CrtW, but still significant (approximately 30%) when the position of genes was inverted. Therefore, the engineered strains constitute a useful tool for investigating the ketocarotenoid biosynthetic pathway in cyanobacteria and an excellent starting point for further optimisation and industrial exploitation of these organisms for the production of added‐value compounds.

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“…A more general platform for high‐throughput functional analysis requires a less specific physiological structure and a shorter timescale than that provided by maize endosperm. Previous studies have shown that callus cultures in species such as maize, Arabidopsis thaliana , sweet potato ( Ipomoea batatas ), marigold ( Tagetes erecta ) and banana ( Musa acuminata ) may be used to test gene function (Paine et al ., ; Harada et al ., ; Kim et al ., , ,b; Vanegas‐Espinoza et al ., ; Mlalazi et al ., ). However these studies have been limited to single genes driven by a constitutive promoter because complex and time‐consuming strategies are required to construct cassettes carrying multiple transgenes.…”

Section: Introductionmentioning

confidence: 99%

An in vitro system for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation

et al. 2014

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SUMMARYWe have developed an assay based on rice embryogenic callus for rapid functional characterization of metabolic genes. We validated the assay using a selection of well-characterized genes with known functions in the carotenoid biosynthesis pathway, allowing rapid visual screening of callus phenotypes based on tissue color. We then used the system to identify the functions of two uncharacterized genes: a chemically synthesized b-carotene ketolase gene optimized for maize codon usage, and a wild-type Arabidopsis thaliana ortholog of the cauliflower , we found that the wild-type Orange allele was sufficient to induce chromoplast differentiation. We also found that chromoplast differentiation was induced by increasing the availability of precursors and thus driving flux through the pathway, even in the absence of Orange. Remarkably, we found that diverse endosperm-specific promoters were highly active in rice callus despite their restricted activity in mature plants. Our callus system provides a unique opportunity to predict the effect of metabolic engineering in complex pathways, and provides a starting point for quantitative modeling and the rational design of engineering strategies using synthetic biology. We discuss the impact of our data on analysis and engineering of the carotenoid biosynthesis pathway.

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Simple functional analysis of key genes involved in astaxanthin biosynthesis using Arabidopsis cultured cells

Cited by 7 publications

References 63 publications

Metabolic engineering of Lilium × formolongi using multiple genes of the carotenoid biosynthesis pathway

Metabolic engineering of Lilium × formolongi using multiple genes of the carotenoid biosynthesis pathway

Non‐endogenous ketocarotenoid accumulation in engineeredSynechocystissp. PCC 6803

An in vitro system for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation

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