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Building Better Carotenoids Three Ways C arotenoids, a family of colorful an- tioxidant natural products, are marketed commercially for a range of industrial and medical applica- tions, although they are not easy to iso- late or synthesize. Three independent research groups have now developed genetic engineering techniques that should make it a whole lot easier to ob- tain known carotenoids in substantial quantities and to create some entirely new ones as well. Carotenoids are essential to microbi- al, plant, and animal life, note postdoc Joseph M. Jez and associate professor Joseph P. Noel of the Structural Biology Laboratory at the Salk Institute for Bio- logical Studies, La Jolla, Calif., in a writ- ten commentary on the three Nature Biotechnology scientific papers that de- scribe the techniques. The compounds act as protective antioxidants in photo- synthetic plants and in organisms— including people—that eat carotenoid- containing foods. As vitamin A precur- sors, they play an essential role in the visual system. They serve as important colorants of flowers, fruits, and vegeta- bles, such as tomatoes and carrots. And they are marketed commercially for use in a variety of cosmetics and foods. SRI Consulting, Menlo Park, Calif., es- timates the 1999 world market for carot- enoids at $750 million to $800 million per year, with Roche and BASF the main glo- bal suppliers. Nevertheless, only a hand- ful of carotenoid compounds can current- ly be synthesized in substantial quanti- ties, extracted from natural sources, or obtained by microbial fermentation. In earlier efforts to increase the avail- ability of carotenoids, one research group used recombinant DNA technology to create β-carotene-containing rice as a means to fight vitamin A deficiency—a serious public health problem in poor countries—and another created trans- genic tomatoes with enhanced carote- noid content. Monsanto, which owns key patents on β-carotene-enriched rice, an- nounced earlier this month that it would allow thericeto be produced on a royalty- free basis as a means to help alleviate vita- min A deficiencies worldwide. Now, three research groups have de- veloped new genetic engineering routes to carotenoid compounds. One group— consisting of assistant professor of bio- chemistry Claudia Schmidt-Dannert and postdoc Daisuke Umeno of the Uni- versity of Minnesota, St. Paul, and pro- fessor of chemical engineering and bio- chemistry Frances H. Arnold of Califor- nia Institute of Technology—used a DNA-shuffling technique to breed bac- teria that produce novel carotenoids [Nat. Biotechnol, 18, 750 (2000)]. The technique involved taking gene frag- ments from different bacterial species and combining them to form a large va- riety of carotenoid genes, which were then expressed in bacterial host cells. One of the resulting products, a carot- enoid called torulene, is not produced by any known bacteria. According to the researchers, the DNA-shuffling ap- proach "may allow the discovery and production, in simple laboratory organ- isms, of new compounds that are essen- tially inaccessible from natural sources or by synthetic chemistry." A related strategy was used by pro- fessor Gerhard Sandmann of the Botan- Top: Artists rendering of bacteria colored by genetic engineering of their carotenoid biosynthetic pathways. Bottom: Colorful extracts from bacterial cells that Schmidt-Dannert, Umeno, and Arnold engineered to produce new carotenoids. ical Institute at Goethe University, Frankfurt, Germany, and coworkers [Nat Biotechnol, 18,843 (2000)].They took nonmodified genes for a variety of carotenoid biosynthetic enzymes from different bacteria and combined them in plasmids, which were then introduced into bacterial host cells. When the mixed genes were coex- pressed in the bacterial host, 12 different carotenoid compounds were produced, four of which had never previously been isolated or synthesized chemically. And the antioxidative activity of these four ca- rotenoids was relatively high, "making them interesting pharmaceutical candi- dates," according to the researchers. Genetics professor Joseph Hirsch- berg and coworkers at the Hebrew Uni- versity of Jerusalem, in Israel, adopted a somewhat different approach to engi- neering of carotenoid biosynthesis by producing the compounds in tobacco plants rather than in bacteria [Nat. Bio- technol, 18, 888 (2000)]. They manipu- lated tobacco's carotenoid biosynthetic pathway by adding a ketolase enzyme from red algae. The modified plant biosynthesized astaxanthin, a compound responsible for the characteristic pink coloring of salm- on, trout, and shrimp. Astaxanthin pro- duced by chemical synthesis is currently marketed commercially for use in salmon farming. However, it is also of interest for potential medical applications because it has been shown to boost immune func- tion in humans, reduce oral cancer in rats, and inhibit breast cancer in mice. The approach developed by Hirsch- berg and coworkers not only provides a way to produce astaxanthin in tobacco but could also be applicable to the pro- duction of novel carotenoids in other types of plants. "It's not clear yet if the biotechnological production of caroten- oids can become cheaper than chemical synthesis," Hirschberg tells C&EN. "However, the biotechnology approach has other advantages: 'cleaner' produc- tion, renewable sources, and the ability to provide natural products," such as specific carotenoid enantiomers. According to Jez and Noel, the three papers "give us a glimpse of what the fu- ture holds for carotenoid biosynthesis in hosts amenable to large-scale fermen- tation and in plants. ... Ultimately, a combination of methodologies is most likely to lead to more diverse libraries of novel carotenoids and a kaleidoscope of opportunities." Stu Borman AUGUST 14,2000 C&EN 39

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Page 1: Building Better Carotenoids Three Ways

Building Better Carotenoids Three Ways

C arotenoids, a family of colorful an­tioxidant natural products, are marketed commercially for a

range of industrial and medical applica­tions, although they are not easy to iso­late or synthesize. Three independent research groups have now developed genetic engineering techniques that should make it a whole lot easier to ob­tain known carotenoids in substantial quantities and to create some entirely new ones as well.

Carotenoids are essential to microbi­al, plant, and animal life, note postdoc Joseph M. Jez and associate professor Joseph P. Noel of the Structural Biology Laboratory at the Salk Institute for Bio­logical Studies, La Jolla, Calif., in a writ­ten commentary on the three Nature Biotechnology scientific papers that de­scribe the techniques. The compounds act as protective antioxidants in photo-synthetic plants and in organisms— including people—that eat carotenoid-containing foods. As vitamin A precur­sors, they play an essential role in the visual system. They serve as important colorants of flowers, fruits, and vegeta­bles, such as tomatoes and carrots. And they are marketed commercially for use in a variety of cosmetics and foods.

SRI Consulting, Menlo Park, Calif., es­timates the 1999 world market for carot­enoids at $750 million to $800 million per year, with Roche and BASF the main glo­bal suppliers. Nevertheless, only a hand­ful of carotenoid compounds can current­ly be synthesized in substantial quanti­ties, extracted from natural sources, or obtained by microbial fermentation.

In earlier efforts to increase the avail­ability of carotenoids, one research group used recombinant DNA technology to create β-carotene-containing rice as a means to fight vitamin A deficiency—a serious public health problem in poor countries—and another created trans­genic tomatoes with enhanced carote­noid content. Monsanto, which owns key patents on β-carotene-enriched rice, an­nounced earlier this month that it would allow the rice to be produced on a royalty-free basis as a means to help alleviate vita­min A deficiencies worldwide.

Now, three research groups have de­veloped new genetic engineering routes to carotenoid compounds. One group— consisting of assistant professor of bio­

chemistry Claudia Schmidt-Dannert and postdoc Daisuke Umeno of the Uni­versity of Minnesota, St. Paul, and pro­fessor of chemical engineering and bio­chemistry Frances H. Arnold of Califor­nia Institute of Technology—used a DNA-shuffling technique to breed bac­teria that produce novel carotenoids [Nat. Biotechnol, 18, 750 (2000)]. The technique involved taking gene frag­ments from different bacterial species and combining them to form a large va­riety of carotenoid genes, which were then expressed in bacterial host cells.

One of the resulting products, a carot­enoid called torulene, is not produced by any known bacteria. According to the researchers, the DNA-shuffling ap­proach "may allow the discovery and production, in simple laboratory organ­isms, of new compounds that are essen­tially inaccessible from natural sources or by synthetic chemistry."

A related strategy was used by pro­fessor Gerhard Sandmann of the Botan-

Top: Artists rendering of bacteria colored by genetic engineering of their carotenoid biosynthetic pathways. Bottom: Colorful extracts from bacterial cells that Schmidt-Dannert, Umeno, and Arnold engineered to produce new carotenoids.

ical Institute at Goethe University, Frankfurt, Germany, and coworkers [Nat Biotechnol, 18,843 (2000)].They took nonmodified genes for a variety of carotenoid biosynthetic enzymes from different bacteria and combined them in plasmids, which were then introduced into bacterial host cells.

When the mixed genes were coex-pressed in the bacterial host, 12 different carotenoid compounds were produced, four of which had never previously been isolated or synthesized chemically. And the antioxidative activity of these four ca­rotenoids was relatively high, "making them interesting pharmaceutical candi­dates," according to the researchers.

Genetics professor Joseph Hirsch-berg and coworkers at the Hebrew Uni­versity of Jerusalem, in Israel, adopted a somewhat different approach to engi­neering of carotenoid biosynthesis by producing the compounds in tobacco plants rather than in bacteria [Nat. Bio­technol, 18, 888 (2000)]. They manipu­lated tobacco's carotenoid biosynthetic pathway by adding a ketolase enzyme from red algae.

The modified plant biosynthesized astaxanthin, a compound responsible for the characteristic pink coloring of salm­on, trout, and shrimp. Astaxanthin pro­duced by chemical synthesis is currently marketed commercially for use in salmon farming. However, it is also of interest for potential medical applications because it has been shown to boost immune func­tion in humans, reduce oral cancer in rats, and inhibit breast cancer in mice.

The approach developed by Hirsch-berg and coworkers not only provides a way to produce astaxanthin in tobacco but could also be applicable to the pro­duction of novel carotenoids in other types of plants. "It's not clear yet if the biotechnological production of caroten­oids can become cheaper than chemical synthesis," Hirschberg tells C&EN. "However, the biotechnology approach has other advantages: 'cleaner' produc­tion, renewable sources, and the ability to provide natural products," such as specific carotenoid enantiomers.

According to Jez and Noel, the three papers "give us a glimpse of what the fu­ture holds for carotenoid biosynthesis in hosts amenable to large-scale fermen­tation and in plants. . . . Ultimately, a combination of methodologies is most likely to lead to more diverse libraries of novel carotenoids and a kaleidoscope of opportunities."

Stu Borman

AUGUST 14,2000 C&EN 3 9