Tuning a Genetic Orchestra


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   (l-r) Lior Zelcbuch, Dr. Ron Milo and Niv Antonovsky




Though the process is highly complex, research on metabolism carries rich rewards: The active molecules produced through plant metabolism, for example, often have considerable benefits when we consume them. Thus for instance, the metabolite lycopene, which gives tomatoes their red color, is thought to help prevent cancer. In certain algae, the metabolic pathway proceeds in succeeding steps from lycopene to beta carotene, the carrot-orange metabolite that is also thought to confer health benefits, and eventually leads to astaxanthin. Astaxanthin is an antioxidant that is today marketed as a health supplement, and it may have a number of potential uses in medicine.


Bacterial colonies that express different metabolites


Dr. Ron Milo and research students Niv Antonovsky and Lior Zelcbuch of the Plant Sciences Department turned their sights on astaxanthin because the algae that produce it are hard to cultivate, and they require special conditions to make the metabolite. For Milo and his group, this was a side project. They are working with the bacterium E. coli to see if they can coax it to absorb carbon dioxide from the atmosphere, as plants do. The scientists use E. coli – the workhorse of the genetic engineering field – because its genome is well known and easy to manipulate. Convincing the bacteria to produce astaxanthin would be a sort of test case for some cutting-edge methods of genetic engineering the scientists are using in experiments.

To get a bacterium to produce an algal metabolite requires more than inserting a gene or two into its genome. The entire network of genes that play in the metabolic “orchestra” must be reprogrammed. This process is referred to as “metabolic engineering.” It is something like planning an industrial process: A multi-stage work plan must be drawn up to encompass the entire assembly line as well as coordinating between the individual stations. Thus, for instance, if one station turns out an intermediate product in amounts that are too great for the next station on the line to handle, there will be a backup in the system. If, on the other hand, a station works too slowly, the rest of the plant will not be able to function at peak efficiency.
In other words, a good deal of fine-tuning is needed. To achieve the best levels of efficiency, the researchers applied genetic engineering techniques to a number of genes at once in the E. coli. From this they obtained thousands of different strains, from which they could select those with the desired properties. To adjust the output of the steps in the metabolic pathway they used a sort of “volume control button” on each gene. This is a segment at the beginning of the gene that gives instructions to the ribosome – the cellular factory that translates the genetic information to proteins. Small changes in the sequences of these segments can lead to significant differences in the intensity of gene expression.
To begin with, the research team generated random mutations in the “volume control buttons” of the genes involved in astaxanthin production and then inserted these genes into E. coli. The bacteria in their lab dishes began to produce engineered enzymes, and these, in turn, produced the different intermediate metabolites on the astaxanthin metabolic pathway.
How did the team identify the strains that produced astaxanthin the most efficiently? Here nature came to the scientists’ aid: Astaxanthin is pink, and thus the bacterial colonies that were the loveliest pink color were those that had the most finely tuned metabolic pathways for its production. The most promising strains then underwent biochemical analysis to quantify astaxanthin levels. The best of these was found to yield over five times as much astaxanthin as other research groups around the world had managed to produce through metabolic engineering in bacteria. These results recently appeared in Nucleic Acids Research.

Milo and his group believe that this method could be used, in the future, in metabolic engineering to improve efficiency in the production of bioactive substances and drugs.
Dr. Ron Milo's research is supported by the Mary and Tom Beck-Canadian Center for Alternative Energy Research; the Lerner Family Plant Science Research Endowment Fund; the European Research Council; the Leona M. and Harry B. Helmsley Charitable Trust; Dana and Yossie Hollander, Israel; the Jacob and Charlotte Lehrman Foundation; the Larson Charitable Foundation; the Wolfson Family Charitable Trust; Charles Rothschild, Brazil; Selmo Nissenbaum, Brazil; the Anthony Stalbow Charitable Trust; and the estate of David Arthur Barton.  Dr. Milo is the incumbent of the Anna and Maurice Boukstein Career Development Chair in Perpetuity.