Bottled enzymes make complex chemicals.

Genetic engineering, which took barely a decade to pervade every biological discipline from biochemistry to systematics, now promises to revolutionize the organic chemistry lab.

With the help of molecular biology, researchers may one day make complex substances in a bottle in much the same way nature creates them in a living celland more efficiently, says A. fan Scott, an organic chemist at Texas A&M University in College Station.

Scott seeks to produce large quantities of substances that organisms typically make slowly in microgram amounts. But like the organisms, he plans to rely on enzymes to do the work for him. Last week, at an American Chemical Society national meeting in Washington, D.C., Scott described his progress using sets of enzymes to make vitamin B12, penicillin, and anticancer alkaloids derived from the rosy periwinkle (SN: 5/30/92, p.366).

"As far as I know, this is the first application of genetic engineering toward trying to understand the biosynthesis of these complicated molecules," says Ronald Breslow, a bioorganic chemist at Columbia University in New York City "There's lots of molecules that organic chemists work on that [this approach] could apply to."

For years chemists have counted on commercially available enzymes to speed chemical reactions. But these researchers typically incorporate just one or two enzymes at key points in a synthesis, or they use enzymes to get a particular starting material, says Mark A. Findeis, a bioorganic chemist at TargeTech, Inc., in Meriden, Conn.

"We're going for the much more difficult enzymes; many of these had been inaccessible until now," says Scott. "And the novelty is putting a lot of these together, like a cocktail."

Scott has spent decades trying to learn how cells make vitamin B12. To do this he had to extract enzymes from living cells. "Often we couldn't get enough to catalyze the reaction," he recalls.

But after others identified the genes that encode the B12-yielding enzymes, Scott began using genetically altered bacteria to mass-produce the enzymes he needs. Molecular biologists insert these genes into bacteria, which then churn out enough enzyme to allow Scott and his colleagues to piece together the pathway's 15 or so steps leading to basic B12.

To determine the order of enzymes in this chemical cascade, Scott and his colleagues use a powerful nuclear magnetic resonance (NMR) spectroscopic technique. By starting with precursor compounds containing a heavy carbon isotope, the researchers can monitor the intermediate products produced.

Thus they can observe whether a particular enzyme modifies a chemical to form the next intermediate along the cascade, Scott explains. Enzymes are quite fussy about the chemicals they work on, typically modifying one intermediate and not others along the cascade. So for each step, the scientists just try each enzyme and monitor with NMR for any chemical changes in that intermediate.

"We've gotten to the point where we can go to the seventh step [in the B12 synthesis]," he adds.

At first Scott's team tried the enzymes one at a time, waiting for each to finish its chemical transformation before adding the next one down the pathway. But one day, when pressed for time, Scott added several enzymes at once to the starting materials. To his surprise, the reaction proceeded smoothly

"Now we put five or six [enzymes] in at a time and leave the bottle for a few hours," he says. He hopes eventually to put all the enzymes involved in B12 synthesis in a single flask at once.

Already he has demonstrated oneflask synthesis with penicillin. This approach cannot compete with commercial production, but it lets researchers try to improve upon this drug by making mutant enzymes and seeing what they produce, Scott says.

In other experiments, he and his colleagues are identifying the periwinkle's enzymes for making medically useful alkaloids. Lacking the genes for these enzymes, the researchers extract a key enzyme, determine its amino acid sequence, and from that sequence reconstruct a gene.

"This kind of effort is requiring the use of a whole passel of enzymes for specific purposes," notes Findeis. "It's beginning to get away from traditional organic chemistry."

That shift is changing how chemists view other disciplines. "Rather than stand apart from molecular biology, we feel it is so much a part of our life now," says Scott. - E. Pennisi