What ever happened to recombinant indigo and designer bluegenes?

Exactly a decade ago, Amgen Inc. scientist Burt D. Ensleystumbled upon a method to genetically engineer indigo fromhost microorganisms.

His paper in Science for Oct. 14, 1983, "Expression ofNaphthalene Oxidation Genes in Escherichia coli Results in theBiosynthesis of Indigo," led to much media coverage at the timeabout "designer genes."

Ensley's U.S. patent, more cryptically titled "MicrobialProduction of Indigo" and assigned to Amgen, issued May 28,1985, as No. 4,520,103. It noted that Adolf von Baeyerannounced the chemical structure of indigo -- C16H10N2O2 --in 1883, and claims: "The present invention provides the firstinstance of microbial production of indigo in a geneticallytransformed microorganism grown in an indole-free medium."

Biosynthetic indigo re-surfaced two weeks ago at the AmericanAssociation for the Advancement of Science (AAAS) meeting inBoston. Alfred L. Gaertner, a senior research scientist atGenencor International, in a talk on "Industrial BiochemicalsThrough Biotechnology," alluded to his company's plans forcommercializing recombinant indigo, which would be moreenvironmentally friendly than indigo produced chemically.Gaertner cited indigo as "the world's largest-selling dye," andpredicted that Genencor would "have a commercial partner inless than a year."

But he also noted that the slow pace of bringing industrialbiotechnology products to the point of sale is due to the smallmarkets they address and to "the chemical industry'sreluctance to adopt new technologies." He quoted a recentcomment in a chemical engineering journal that geneticallyengineered indigo "could never compete with 100 years ofchemical synthesis."

"The problem with genetically engineered indigo is that there isno way it can produce or maintain the capacity required byindustry," Ronald R. Rollek, technical services representative ofthe indigo-manufacturing company Buffalo Color Corp., toldBioWorld.

"It's probably just a nice little thing, tucked in a corner, sayingthat 'yeah, bugs can make indigo,' " Rolleck said. "Beyond that, Idon't believe anybody is really taking it seriously as acompetitive means of producing indigo, versus its presentmethodology. There's no way it can produce or maintain yield."

Buffalo Color Corp. of Buffalo, N.Y., is the sole manufacturer ofindigo dyestuff in the U.S. "Ninety-nine-and-a-half percent ofthe indigo we sell is for the blue-jeans trade," Rollek said.Rough estimates put the world market for indigo at 12,000 tonsa year, at an average $15 or $16 per kilogram, with the U.S.market accounting for 20 percent.

In his AAAS talk, Gaertner described the "pathwayengineering" by which Genencor is scaling up Ensley's bench-top recombinant-indigo process. He confirmed that hiscompany acquired the exclusive license to the invention fromAmgen, and that talks with an unnamed commercial partnerare ongoing. He told BioWorld that, so far, scale-up has reachedthe point of producing recombinant indigo in 55-gallon drumsfor testing and evaluation.

"It's a pretty good process, but until we have tank-car lots, it'snot worth a helluva lot," said Genencor's vice-president forcommunications and public relations, Edward Robinson. He saidhis company has been giving "denim and blue-jean makerssamples of materials we produced."

Ensley, now director of advanced technology at Envirogen Inc.of Princeton, N.J., recalled how he and his fine-chemicals R&Dgroup at Amgen stumbled on recombinant indigo 10 years ago:

"We were interested in using metabolic pathways in bacteria tocatalyze conversion of feedstocks to a variety of high-valuespecialty chemicals," he told BioWorld. "Bacteria that grew onnaphthalene would make intermediates, so I cloned the genesencoding its enzyme, naphthalene dioxygenase, from thenaphthalene degradative pathway of Pseudomonas putida, asoil bacterium, into Escherichia coli. When the E. coli turnedblue, that was unexpected. It made us wonder what we haddone."

In E. coli, but not in P. putida, Ensley explained, there is anenzyme that cleaves tryptophane to indole. Indole is asubstrate for the naphthalene dioxygenase enzyme. Combiningtheir enzymatic pathways lets E. coli synthesize indole, whichthe microbe oxidizes to indigo, and excretes to the surroundingmedium.

Ensley echoed what Gaertner had said at the AAAS meeting,that the current chemical synthesis of indigo "is veryunfriendly environmentally." It involves the disposal of suchhazardous wastes as caustic sodium amide (sodamide), whichforms explosive products when exposed to air. This cleanupfactor, Ensley suggested, is an added industrial cost that makesrecombinant indigo more competitive.

A second Ensley/Amgen/Genencor patent, No. 5,173,425, wasjust issued, titled "Enhancement of Naphthalene DioxygenaseActivity During Microbial Indigo Production." By dint of proteinengineering, Ensley explained, it increases the useful half-lifeof the enzyme by an order of magnitude, to 15 or 20 hours.

Genencor, Gaertner stated, has "a world monopoly" ongenetically engineered indigo. But W.R Grace & Co. may beplanning a biological end-run. Just over a year ago, it won U.S.patent No. 5,077,201, which lists 18 claims for the use of amutated fungal culture to produce indigo.

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.

No Comments