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Noda Institute for Scientific Research GRANT2013 Research Grant

Construction of an efficient system for the production of redpepper carotenoids and new derivativesNorihiko MisawaIshikawa Prefectural University, Research Institute for Bioresources and Biotechnology

Research aimsCapsanthin and capsorubin are useful functional carotenoids with a strong capability of decreasing singlet oxygen. Since they are found only in the fruit of red pepper orpollen grains of the lily, it is difficult to extract them inlarge amounts. 4-Ketocapsanthin is a novel carotenoid,which has a chemical structure of both capsanthin andastaxanthin. Astaxanthin is used as an anti-oxidant in foods.
Therefore, 4-ketocapsanthin is expected to be a ‘super antioxidant’. We have constructed pathway-engineered Escherichia coli cells that can effectively produce an excess offarnesyl pyrophosphate (FPP).1) In this study, we propose touse this E. coli to develop a system for the production ofcapsanthin, capsorubin, or the novel carotenoid, 4-ketocapsanthin. For this purpose, we will clone and introduce thegenes required for the biosynthesis of these carotenoids.
The goal of our study is to facilitate a comprehensive examination of the biological activities of these pigments.

Fig. 1. Analysis of products from genetic engineered E. coli.
(A) HPLC chromatogram of the recombinant E. coli M12 (pET-MpZEP),(B) HPLC chromatogram ot the recombinant E. coli M12 (pET-MpZEP/GDH+pRSF-ferredoxin/FNR), (C) Absorption spectra of individual peaks.
1, zeaxanthin; 2, novel carotenoid.

Methods

Results

1. Effective production of zeaxanthinFor the efficient production of zeaxanthin (over 10 mg/gDW), we have constructed an E. coli strain expressing thecrtE, crtB, crtI, crtY, and crtZ genes from the soil bacterium Pantoea ananatis as well as the Haematococcus pluvialis gene idi (encoding for IPP isomerase).2)1.

2. Effective production of antheraxanthin and violaxanthinTo produce antheraxanthin and violaxanthin in the E.
coli, we introduced the plant zeaxanthin epoxidase (Zep)gene into zeaxanthin-producing E. coli.
3.

Effective production of capsanthin and capsorubinTo produce capsanthin and capsorubin in the E. coli, wewill introduce the capsanthin-capsorubin synthase (Ccs)gene from Capsicum annuum or Lilium lancifolium into theantheraxanthin- and violaxanthin-producing E. coli.
4. Effective production of 4-ketocapsanthinTo produce 4-ketocapsanthin in the E. coli, we will introduce the crtW (carotenoid 4,4ʹ-ketolase) gene from marinebacterium Brevundimonas sp. strain SD212 into the capsanthin- and capsorubin-producing E. coli.

Effective production of zeaxanthinWe constructed the plasmid pACHP-Zea, which containsthe crtE, crtB, crtI, crtY, and crtZ genes from Pantoeaananatis along with the idi gene from Haematococcus pluvialis, and introduced it into E. coli BL21 (DE3) cells. Theproductivity of these recombinant E. coli was 2–3 foldhigher than that of recombinant E. coli bearing the crtE,crtB, crtI, crtY, and crtZ genes but not the idi gene (plasmidpACCAR25ΔcrtX.3) The zeaxanthin-producing E. colistrain carrying the plasmid pACHP-Zea is called M12.
2. Effective production of antheraxanthin and violaxanthinWe cloned the zeaxanthin epoxidase gene (MpZEP) fromliverwort Marchantia polymorpha, to construct the plasmidpET-MpZEP. This plasmid was introduced into the E. coliM12 strain. Neither antheraxanthin nor violaxanthin werepresent in this E. coli M12 (pET-MpZEP) strain. Previousreports demonstrate that functional zeaxanthin epoxidaserequires O2, ferredoxin, ferredoxin reductase (FNR), FAD,and NADPH. Since E. coli are known to lack these co-factors, except for O2, we cloned and introduced the genes expressing ferredoxin-FNR and an NADPH-recycling enzyme (glucose dehydrogenase; GDH) into the recombinant44E. coli M12 (pET-MpZEP) strain. HPLC analysis could detect neither antheraxanthin nor violaxanthin in the resultantrecombinant E. coli M12 (pET-MpZEP/GDH+pRSF-ferredoxin/FNR) strain. Instead, a peak representing a novel carotenoid was found (Fig. 1). Currently, we are in the process of preparing this compound for NMR analysis.
3.

Effective production of capsanthin and capsorubinWe have cloned the capsanthin-capsorubin synthase(Ccs) gene from Capsicum annuum. After the constructionof antheraxanthin- and violaxanthin-producing E. coli, wepropose to introduce the Ccs gene in this strain.
4.

Effective production of 4-ketocapsanthinWe have cloned the crtW (carotenoid 4,4ʹ-ketolase) genefrom the Brevundimonas SD212 strain. After the construction of capsanthin- and capsorubin-producing E. coli, wewill introduce the crtW gene in this strain.
ConclusionsIn this study, we have constructed a recombinant E. colistrain that can efficiently produce zeaxanthin. The combi-nation of zeaxanthin epoxidase, ferredoxin-FNR, andNADPH-recycling led to the unexpected production of anovel carotenoid, although antheraxanthin and violaxanthinwere not detected. Structural identification of the novel carotenoid using NMR analysis is expected to provide insightinto its biosynthesis.
The production of 4-ketocapsanthin from the cloned Ccsand crtW genes will be successfully achieved in the E. coli,after we carry out the production of antheraxanthin and violaxanthin.
References1)

Harada, H., Yu, F., Okamoto, S., Kuzuyama, T., Utsumi, R., and Misawa, N., Efficient synthesis of functional isoprenoids from acetoacetate through metabolic pathway-engineered Escherichia coli. Appl.
Microbiol. Biotechnol., 81: 915–925, 2009.
2) Kajiwara, S., Fraser, P. D., Kondo, K., and Misawa, N., Expressionof an exogenous isopentenyl diphosphate isomerase gene enhancesisoprenoid biosynthesis in Escherichia coli. Biochem. J., 324: 421–426, 1997.
3) Misawa, N., Satomi, Y., Kondo, K., Yokoyama, A., Kajiwara, S.,Saito, T., Ohtani, T., and Miki, W., Structure and functional analysisof a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level. J. Bacteriol.,177: 6575–6584, 1995.