Production of beta-carotene in Zymomonas mobilis and Agrobacterium tumefaciens by introduction of the biosynthesis genes from Erwinia uredovora.

N Misawa, S Yamano and H Ikenaga Appl. Environ. Microbiol. 1991, 57(6):1847.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1991,

p. 1847-1849

Vol. 57, No. 6

0099-2240/91/061847-03$02.00/0 Copyright 1991, American Society for Microbiology

Production of 1-Carotene in Zymomonas mobilis and Agrobacterium tumefaciens by Introduction of the Biosynthesis Genes from Erwinia uredovora
NORIHIKO MISAWA,* SHIGEYUKI YAMANO, AND HIROSHI IKENAGA Central Laboratories for Key Technology, Kirin Brewery Co., Ltd., Gunma 370-12, Japan
Received 24 October 1990/Accepted 20 February 1991

The Erwinia uredovora crtB, crtE, crtl, and crtY genes required for ,8-carotene biosynthesis were introduced by conjugal transfer into an ethanol-producing bacterium, Zymomonas mobilis, and a phytopathogenic bacterium, Agrobacterium tumefaciens, in which no carotenoid is synthesized. The transconjugants of Z. mobilis and A. tumefaciens carrying these genes appeared as yellow colonies and produced 220 and 350 ,ug of ,8-carotene per g of dry weight, respectively, in the stationary phase in liquid culture.

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1-Carotene functions as a precursor of vitamin A in mammals (16) and as a pigment which protects higher plants against photooxidative damage (3, 7). In recent years, this pigment has also attracted attention as one of the nutritional factors for cancer prevention (8). In spite of such important functions, the genes which mediate the biosynthesis of 1-carotene are unknown. This is due to the fact that the carotenogenic enzymes are immediately inactivated by separation from a membrane environment, thus preventing their purification (1, 3) and the subsequent cloning of the genes

pressed (10). Geranylgeranyl PP1 should exist in the cells of many organisms, since it is synthesized by dimethylallyltransferase, a common enzyme in the early steps of the biosynthesis of sterols and terpenes as well as carotenoids (15). It is therefore feasible that our carotenoid biosynthesis genes are capable of producing carotenoids in many cells which are not able to synthesize carotenoids. With this in mind, we introduced the crtB, crtE, crtI, and crtY genes required for 13-carotene synthesis into an ethanol-producing bacterium, Zymomonas mobilis, and into a phytopathogenic

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FIG. 1. Construction of hybrid plasmids pZACAR16 and pBICAR16. Cmr and Kmr, chloramphenicol and kanamycin resistance genes; P, E. uredovora promoter from which transcriptional read-through is considered to occur into crtE, crt Y, crtl, and crtB genes; ori(E) and ori(Z), regions required for replication in E. coli and Z. mobilis, respectively; PCaMv and Tn, CaMV 35S promoter and the nos terminator, which are necessary for transcription in higher plants. In pBICAR16, the carotenoid genes have the opposite orientation from the CaMV 35S promoter and the nos terminator.

encoding them. We have succeeded in cloning the genes for the biosynthesis of cyclic carotenoids containing 1-carotene from a phytopathogenic bacterium, Erwinia uredovora 20D3 (ATCC 19321), as a cluster composed of six structural genes (10). The biosynthetic pathway from geranylgeranyl PP1 to zeaxanthin-13-diglucoside in this bacterium was elucidated by analyzing carotenoids accumulated in Escherichia coli transformants in which some of these six genes were ex*

Corresponding author.
1847

bacterium, Agrobacterium tumefaciens, in which no carotenoid is synthesized, and found that these bacterial strains carrying the biosynthesis genes produced 1-carotene successfully. The plasmid pCAR16delB, carrying crtE, crtY, crtI, and crtB required for 13-carotene biosynthesis (10), was digested with Asp-718 (KpnI)-EcoRI; a 6.0-kb Asp-718-EcoRI fragment containing these four genes was isolated and treated with Klenow enzyme. This fragment was inserted into the EcoRV site of the cloning vector pZA22 for Z. mobilis (11,

1848

NOTES

APPL. ENVIRON. MICROBIOL.

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FIG. 2. Pigmentation of E. coli, Z. mobilis, and A. tumefaciens strains carrying the p-carotene biosynthesis genes of E. uredovora. (A) E. coli JM109(pUC19) (21); (B) E. coli JM109(pCAR16delB) on an LB agar plate containing ampicillin; (C) Z. mobilis NRRL B-14023(pZA22); (D) Z. mobilis NRRL B-14023(pZACAR16) on an RM agar plate containing chloramphenicol; (E) A. tumefaciens C58ClRif'(pBI121); (F) A. tumefaciens C58ClRif(pBICAR16) on an LB agar plate containing kanamycin.

13), creating the hybrid plasmid pZACAR16 (Fig. 1). The 6.0-kb fragment was also ligated to a large filled-in SmaI-SstI fragment, thereby removing the ,-glucuronidase gene from the binary vector pBI121 (Clontech Laboratories, Inc.), which is capable of replicating in A. tumefaciens, creating the hybrid plasmid pBICAR16 (Fig. 1). Hybrid plasmids pZACAR16 and pBICAR16 were introduced into Z. mobilis NRRL B-14023 and A. tumefaciens C58ClRif (5), respectively, by conjugal transfer with the helper plasmid pRK2013 (19, 20). The Zymomonas and Agrobacterium transconjugants appeared as yellow colonies on RM agar plates (Z. mobilis) composed of 1% yeast extract (Oxoid Ltd.), 2% glucose, 0.2% KH2PO4, and 1.5% agar (18) containing chloramphenicol (100 ,ug/ml) and nalidixic acid (100 ,ug/ml) and on Luria broth (LB) agar plates (A. tumefaciens) composed of 1% tryptone (Difco Laboratories), 0.5% yeast extract (Difco), 1% NaCl, and 1.5% agar (9) containing kanamycin (50 ,ug/ml) and rifampin (100 ,ug/ml). These yellow transconjugants are shown in Fig. 2. Plasmid DNAs were prepared from these Zymomonas and Agrobacterium transconjugants by the alkali method (2), subjected to restriction enzyme analysis (9), and found to be maintained stably in these strains. Z. mobilis(pZACAR16) was grown at 30C in T medium composed of 1% yeast extract, 10% glucose, 1% KH2PO4, 1% (NH4)2SO4, and 0.05% MgSO4 7H20 containing chloramphenicol. A. tumefaciens(pBICAR16) was grown at 28C in LB medium containing kanamycin. A yellow pigment was extracted from cells of the stationary phase in the dark with cold acetone. Hexane was added to the acetone extract, and water was added until two phases separated. The lower aqueous phase was removed, and the upper yellow solvent layer was washed three more times with water. The hexane layer was evaporated to dryness. The yellow pigment obtained was dissolved in 2-propanol and subjected to highpressure liquid chromatography; the column (3.9 by 300 mm; Nova-pak HR 6,u C18 [Waters]) was developed with acetonitrile-methanol-2-propanol (90:6:4) (17). UV-visible spectra of elution peaks were measured with a Waters M-490 multiwavelength detector. As a result, the yellow pigments accumulated by Z. mobilis(pZACAR16) and by A. tume-

faciens(pBICAR16) proved to be identical to n-carotene (all trans-P,B-carotene), with a flow rate of 1 ml/min and a retention time of 62 min, by using the commercial authentic sample (Sigma Chemical Co.). Figure 3 shows the production rate of p-carotene in these Zymomonas and Agrobacterium transconjugants and the growth curves. The synthesis of n-carotene by these strains almost appears to be correlated with the number of cells. In the 28 h of cultivation corresponding to the stationary phase, Z. mobilis (pZACAR16) and A. tumefaciens(pBICAR16) produced 280 ,ug of n-carotene per liter of culture (220 jig/g of dry weight)

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FIG. 3. Growth curves of Z. mobilis and A. tumefaciens transconjugants and P-carotene production by these strains. Symbols: O, Z. mobilis NRRL B-14023(pZACAR16); 0, A. tumefaciens

C58ClRifP(pBICAR16).

VOL. 57, 1991

NOTES

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and 490 jig of ,-carotene per liter (350 ,ug/g of dry weight), respectively. In pBICAR16, the 1-carotene biosynthesis genes are inserted with the opposite orientation to that in the cauliflower mosaic virus (CaMV) 35S promoter (14). We also constructed a hybrid plasmid carrying these genes in the same orientation as in the CaMV 35S promoter, but this hybrid plasmid was not introduced into A. tumefaciens. It is generally true that a foreign gene inserted with the same orientation as in the CaMV 35S promoter in pBI121 is also well expressed in A. tumefaciens. It is therefore likely that ,B-carotene production at a higher level in this bacterium may lead to the death of cells, presumably by disturbing a membrane environment. No attempt to extend the range of utilizable sugars by genetic manipulation of Z. mobilis has resulted in success, although some foreign genes which encode sugar-degrading enzymes have been expressed in this microorganism (4, 6, 12). The Z. mobilis cells accumulating 1-carotene may be able to be utilized as nutrients for farm animals after carrying out ethanol production. This means that there is another means of extending the sugar-utilizing ability, in order to exploit the potential of this bacterium for its commercial use in alcoholic fermentation.
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