Biopharmconsortium Blog

2010

Breakthroughs of the Year

As it does every year, Science published its “Breakthrough of the Year” for 2010 in the 17 December issue of the journal.

For its “Breakthrough of the Year”, Science chose a non-life science innovation, the first quantum machine. Interestingly, the same issue of Science included a Perspective on biophysicist Britton Chance, who died last November at the age of 97. Among his many accomplishments, Dr. Chance discovered that biological electron transfer operates via quantum tunneling, a mechanism central to photosynthesis, respiration, and many oxidoreductase enzymes. Mitochondria, chloroplasts, and oxidoreductase enzymes thus constitute biological quantum machines of a sort.

Interestingly, Dr. Chance continued to work on metabolism all of his long working life, including in the era of molecular biology when interest in that field waned. By doing so, he made many important contributions, including the mechanism for the generation of the reactive oxygen species (ROS) superoxide and peroxide during normal mitochondrial respiration, and the  use of near-infrared (NIR) light for noninvasive diagnostics.

Although Science chose a non-life science advancement as its “Breakthrough of the Year”, the journal’s runners-up for “Breakthrough of the Year” were replete with life science items. The first runner-up was the synthetic Mycoplasma mycoides genome constructed by the J. Craig Venter Institute, which they used to create “the first synthetic cell”. As we discussed in a series of two articles on this blog (see here and here, although the creation of the synthetic genome and the “synthetic cell” represented a technical tour de force, it did not represent a true breakthrough. Many leading scientists, including leaders in the field of synthetic biology, agreed with us. However, at least several bioethicists and philosophers hailed this work as a milestone, calling it “the end of vitalism”. (As we noted in another blog post, however, not all bioethicists agree.)

Moreover, policy-makers were sufficiently alarmed by the “synthetic cell” that (as noted in the Science “Breakthrough of the Year” runners-up article) the Presidential Commission for the Study of Bioethical Issues held hearings on policy implications of this research. Nevertheless, the report of this commission (issued in December 2010) concluded that the Venter research “does not amount to creating life as either a scientific or a moral matter” and that synthetic biology remains “in the early stages,” with any dangers well into the future. The commission recommended continuing White House oversight, but a relatively mild set of regulatory measures.

As we said in our second article on the “synthetic cell”, we are much more impressed by the metabolic engineering studies of Jay Keasling, and by George Church’s automated method for optimizing metabolic engineering pathways, which we had discussed in an earlier blog post. The Science “Breakthrough of the Year” runners-up article mentioned Dr. Church’s automated system, among other synthetic biology advances made in 2009 and 2010.

Meanwhile, in a review of metabolic engineering published in the 3 December 2010 issue of Science, Dr. Keasling says that although minimal bacterial hosts such as Dr. Venter’s “synthetic” mycoplasma may be of scientific interest, they are not suitable to use in metabolic engineering studies whose goal is scale-up for industrial production of medicines, chemicals, or biofuels. This agrees with our statement that such applications require  “workhorse” organisms that can take the extensive genetic manipulation needed to engineer new metabolic pathways, and which are capable of scale-up.

We therefore believe that the “synthetic cell” is not the life science breakthrough of the year, despite its placement at the top of Science‘s “Breakthrough of the Year” runners-up article.

Our nominee for the life science breakthrough of the year is listed right under the “synthetic cell” in the Science “Breakthrough of the Year” runners-up article. It is the determination of the sequence of approximately two-thirds of the Neanderthal genome by Svante Pääbo (Max-Planck Institute for Evolutionary Anthropology, Leipzig, Germany.) and his colleagues. This achievement is something that only a few years ago seemed completely impossible. Moreover, this work is of great cultural significance, since it indicates that Neanderthals contributed some 1-4 percent of the genome sequences of non-African present-day humans. More recently, Dr. Pääbo and his colleagues followed up the Neanderthal studies by using their DNA synthesis methods to identify a third species of humans, known as Denisovans. Denisovans, who were more closely related to Neanderthals than to modern humans, were alive at the same time as modern humans emerged from Africa and also encountered the Neanderthals. Dr. Pääbo’s new studies indicate that the Denisovans contributed some 4–6% of the genome sequences of present-day Melanesians.

Despite the importance of the Pääbo Neanderthal studies, we have not blogged on this work simply because it has nothing to do with drug discovery and development. However, perhaps someday, for example, some of the products of genes that are found in present-day humans but not in Neanderthals could emerge as potential drug targets. As discussed in the Science “Breakthrough of the Year” runners-up article, researchers have begun studying some of these gene products in cell culture systems.

Moreover, the types of advanced, next-generation DNA sequencing methods used by Dr. Pääbo and his colleagues are being applied to studies that are relevant to drug discovery. These include the 1000 Genomes Project, which seeks to find all single-nucleotide polymorphisms (SNPs) present in at least 1% of humans. This and other next-generation genomics projects were listed in the Science “Breakthrough of the Year” runners-up article, as the third runner-up. The 1000 Genomes Project, as well as genome-wide association studies (GWAS) that use high-throughput DNA sequencing methods, may enable researchers to identify rare mutations that are involved in complex human diseases. This might in turn lead to the discovery of novel drugs and diagnostics.

Among other life science items in the Science “Breakthrough of the Year” runners-up article was the production of knockout rats. We discussed knockout rats in an October 1, 2010 blog post.

Newsmaker of the Year

Nature also had an end-of-2010 special article, “The Newsmaker of the Year”, in its 23/30 December issue. Unfortunately, Nature chose a U.S. government official as its Newsmaker of the Year.

We would prefer that Nature stick to what it does so very well, and stay out of U.S. politics, whether in its “opinionated editorials” [sic] or elsewhere. Perhaps the low point in Nature‘s political forays was its November 2010 editorial calling for what amounts to a new version of Prohibition. This is despite the ample evidence that moderate consumption of red wine (for example) is healthy for most adults. Readers would be well advised not to believe everything they read in Nature editorials.

Our nominee for Newsmaker of the Year in the life sciences is Dr. Svante Pääbo, for the reasons we discussed earlier.

Deals of the Year

Also as an end-of-year feature, the IN VIVO blog has been running a Deal of the Year competition. The nominees are grouped in three categories: M&A Deal of the Year, Alliance Deal of the Year, and Exit/Financing Deal of the Year.

Only one of the nominees had been featured in an article on our blog: the Celgene/Agios alliance (April 23, 2010).

The IN VIVO Blog invited readers to vote on the Deal of the Year in each of the three categories, by going to http://www.windhover.com/ezine/html/doty10.html. The voting closed at 12:00pm on 6 January 2011 (Eastern Standard Time).

The winners of the vote were:

• M&A Deal of the Year: Celgene/Abraxis (50.31% of 1,799 votes)
• Alliance Deal of the Year: Celgene/Agios (55.32% of 3,176 votes)
• Exit/Financing Deal of the Year: Ablexis (46.54% of 1,631 votes)

Congratulations to all the winners, especially Agios and Celgene, which were featured in our blog post.

Happy New Year!

This is our own version of an end-of-year special article, and will be our last blog post of 2010. Best wishes to all of you for a happy, productive, and innovative New Year in 2011.

As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

Citric acid cycle

The 3 December issue of Science featured a Special Section on metabolism, headed by an introductory article entitled “Metabolism is Not Boring”.

Way back in the 1920s through the 1950s, intermediary metabolism was a hot field of biology. This culminated in the awarding of the Nobel Prize in Physiology or Medicine in 1953 to Hans Krebs “for his discovery of the citric acid cycle” and Fritz Lipmann “for his discovery of co-enzyme A and its importance for intermediary metabolism”.

As most of you know, that same year, 1953, Watson and Crick published the structure of DNA, which won them the Nobel Prize in Physiology or Medicine in 1962. This began the great era of molecular biology. As the result of the overwhelming success of molecular biology, the study of intermediary metabolism receded into the background. Answering most questions in leading-edge biology required little or no attention to intermediary metabolism. However, as discussed in the review article by Steven L. McKnight included in the Special Section, metabolism is coming to the forefront of biomedicine again. Research problems that require both consideration of molecular biology and of metabolism now appear as interesting and important challenges.

Considerations of intermediary metabolism have always been important in the study of what are known as metabolic diseases, especially type 2 diabetes and obesity and such related conditions as dyslipidemia. However, as detailed both in the McKnight article and in an article by Arnold J. Levine and Anna M. Puzio-Kuter, the study of intermediary metabolism has now also become important in cancer, with the discovery that alterations in metabolic enzymes can result in the production of “oncometabolites” that support the growth of cancer cells.

In an article on this blog dated December 31, 2009, we discussed research in cancer metabolism that is behind the technology platform of Agios Pharmaceuticals (Cambridge, MA). In that article, we highlighted the discovery that mutations in a metabolic enzyme, cytosolic isocitrate dehydrogenase (IDH1) are a causative factor in a major subset of human brain cancers. The wild-type form of IDH1 catalyzes the NADP+-dependent oxidative decarboxylation of isocitrate to α-ketoglutarate. However, the mutant forms of IDH1 catalyzes the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG). 2HG appears to be an oncometabolite that is involved in the progression of low-grade gliomas to lethal secondary glioblastomas. Agios researchers and their academic collaborators later implicated mutations in isocitrate dehydrogenase enzymes and the production of the oncometabolite 2HG in the pathogenesis of acute myelogenous leukemia (AML).

Also discussed in our article was the Warburg effect, in which cancer cells carry out aerobic glycolysis (conversion of glucose to lactate, with the production of 2 molecules of ATP even in the presence of oxygen). In contrast, most normal mammalian cells metabolize glucose to CO2 and water via glycolysis coupled to the mitochondrial citric acid cycle, generating 36 molecules of ATP. Agios scientific founder and signal-transduction pioneer Lewis Cantley showed that there is a connection between growth factor-mediated signal transduction and aerobic glycolysis in cancer cells. In particular, Dr. Cantley and his colleagues found that pyruvate kinase M2 (PKM2) is a link between signal transduction and aerobic glycolysis. PKM2 binds to tyrosine-phosphorylated signaling proteins, which results in the diversion of glycolytic metabolites from energy production via mitochondrial oxidative phosphorylation to anabolic processes required for rapid proliferation of cancer cells.

The McKnight and Levine and Puzio-Kuter papers also discuss the Warburg effect in cancer cells, and the role of mutations in several metabolic enzymes that contribute to malignant phenotypes. The McKnight article notes that in addition to dominant mutations in isocitrate dehydrogenates, rare recessive mutations in fumarate hydratase and succinate dehydrogenase are also associated with cancer. Mutations in the genes for these enzymes, coupled with loss of the wild-type allele, result in elevated intracellular levels of fumarate and succinate, respectively. These appear to act as oncometabolites that can induce activation of the hypoxia response pathway, which triggers the induction of aerobic glycolysis (the Warburg effect) and angiogenesis.

The Levine and Puzio-Kuter paper also discusses the role of oncogenes and tumor suppressor genes and their signaling pathways in regulating metabolism and in particular in inducing the Warburg effect. For example, p53 regulation suppresses the Warburg effect and promotes mitochondrial oxidative metabolism. Thus the loss of p53 function seen in most human cancers tends to promote aerobic glycolysis. Other signaling pathways that have been implicated in cancer-associated changes in metabolism include the Akt and mTOR pathways, which are frequently altered by mutations in key genes (e.g., mutations in PTEN and amplifications of such growth factor receptors as Her2 and EGFR) in cancer.  Deregulation of these pathways activates the hypoxia response pathway, thus triggering the Warburg effect.

Levine and Puzio-Kuter suggest that research aimed at a deeper understanding of how cancer-associated signaling pathways regulate biochemical metabolic pathways and trigger the Warburg effect, and the role of the Warburg effect in the pathogenesis of cancer, may lead to novel drug discovery strategies in oncology.

The Special Section on metabolism also includes an article on autophagy, a process by which cells break down cellular components in order to eliminate damaged biomolecules and organelles or to provide substrates for metabolism in case of starvation. Although autophagy promotes the health of cells and can prevent degenerative diseases, it can also enable cancer cells to survive in nutrient poor tumors.

There is also a review by Jay Keasling on metabolic engineering to produce such substances as natural product drugs, chemicals, and biofuels. Metabolic engineering is a branch of synthetic biology that engineers metabolic pathways to produce such substances, hence the inclusion of this review in the Special Section on metabolism. We have several articles on synthetic biology on this blog, most of which focus on metabolic engineering and its role in drug manufacture and drug discovery.

All in all, the 3 December Special Section on metabolism is worth reading by basic researchers, and by drug discovery and development researchers in biotechnology and pharmaceutical companies. It may broaden your perspectives, and lead to new ideas for R&D or partnering, especially in oncology drug discovery.
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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

In our last two blog posts–dated July 12, 2010 and July 18, 2010, we discussed the “synthetic cell” that was recently constructed by researchers at the J. Craig Venter institute. As we discussed, at least several leading bioethicists and philosophers said that the construction of a “synthetic” microbial cell refuted vitalism–i.e., the contention that there is something special about processes in living organisms that cannot be artificially created from nonliving systems–once and for all. However, leading scientists (including Nobel Prize winners and leading synthetic biologists) disagreed with that assessment. We said that we agreed with the leading scientists, and gave our reasons why.

Meanwhile, a one-page essay by bioethicist Gregory Kaebnick, Ph.D. appeared In the July 2010 issue of The Scientist (registration required).  Dr. Kaebnick is the editor of the bioethics journal The Hastings Center Report, and a co-investigator of a Hastings Center research project on synthetic biology. Dr. Kaebnick agrees with the leading scientists, and with us, even though his friend and colleague Arthur Caplan is one of the bioethicists who says that the “synthetic cell” has refuted vitalism.

According to Dr. Kaebnick, what the Venter group created was not a synthetic cell, but a synthetic genome. (As we stated in our second article, the researchers had help from yeast in creating the “synthetic” genome–perhaps it is really a semi-synthetic genome.) But the Venter group says that since the genome took over the cell it was transferred into, and since the genome is synthetic, therefore the cell is synthetic. But that assumes a top-down control of a cell by its genome (i.e., genetic determinism). Dr. Kaebnick argues that one might instead say that the cell and the genome worked out their differences and collaborated, or that the cell “adopted” the genome. He goes on to assert that we may not know enough to say which of these two metaphors is most adequate.

Then Dr. Kaebnick goes on to ask whether even if the Venter group did create a synthetic cell, whether that really demystified life at all. You will have to read his article to follow that argument.

From our point of view, even if the “top down” control model is the most nearly correct, without a pre-formed cell it would have been impossible to use the synthetic genome to create a living organism. Researchers cannot, at least at present, create a cell, with its membranes, organization of biomolecules, biochemical systems, etc. that is necessary for a genome to work to express itself in a living system.

Moreover, with the discoveries on epigenetics in the last decade or so, researchers know that a “top down” control model–especially in multicellular eukaryotic organisms–does not fully account for how cells and organisms work. The environment can mediate changes in chromatin, such as DNA methylation and histone modification, which can be passed down from cell to cell and in some cases even to the next generation.

Thus the issue of “top down” genetic determinism versus collaboration between a cell and its genome has implications for cutting-edge biological research. Since some drug discovery researchers have been working on discovery and development of epigenetics-based drugs, it is of interest to the biotechnology/pharmaceutical industry as well. Several such drugs, including Celgene’s DNA methyltransferase inhibitors and histone deacetylase inhibitors that we mentioned in an earlier blog post, are already on the market.

In our previous (July 12) blog post, we began a discussion of the creation of the “synthetic cell” by J. Craig Venter and his colleague at the J. Craig Venter Institute (JCVI).  In this study, the researchers produced a synthetic version of the genome of bacterium Mycoplasma mycoides. They then transferred the synthetic M. mycoides genome into the closely related bacterium M. capricolum, where then new genome took over the cells, resulting in bacteria that expressed the proteome of M. mycoides. The resulting cells were dubbed “synthetic cells”.

We said that we agreed with scientists (including working synthetic biologists) who said that the “synthetic cell” project did very little to advance synthetic biology, beyond the demonstration of technical virtuosity. Here’s why we agree.

JCVI’s “synthetic genome” is merely a copy of the natural M. mycoides genome, with a few minor differences. Copying the natural genome does not advance our understanding of how the various genes of M. mycoides function. Even in the case of such a simple organism as M. mycoides, only about half the genes in the genome are understood in terms of their biological function.

Copying the natural genome of M. mycoides also does not tell us how the genes of this organism work together in biological processes. Researchers find it difficult to construct even simple synthetic biology devices, because it is difficult to get artificially-introduced genes to work together as planned even in prokaryotic systems. For example, in 2000 researchers constructed a simple biological clock based on a three-gene “repressilator network” in E. coli. The clock was designed so that the cells flash on and off, based on oscillating expression of a fluorescent protein. However, the operation of this simple clock was “noisy”; i.e., the period of these oscillations was highly variable. However, natural biological clocks are not noisy. Other researchers therefore designed a more complex synthetic clock to damp down the noise of the original clock. This example illustrates how the complexity of biology can affect the ability of researchers to construct even simple biological devices. However, constructing such devices and then working to improve them can help researchers gain a greater understanding of how genes work together in natural networks and processes.

Every year since 2004, there has been an international undergraduate synthetic biology competition, run out of MIT, called the International Genetically Engineered Machine (iGEM) competition. In this competition, students use “biological parts” from a “kit”, and/or develop new “parts”, to design a synthetic biology device. Many teams that participate in this competition find out that designing new synthetic biology systems is not a simple case of putting together “biological parts” like Lego blocks or components of an electrical circuit. Not all the “parts” deposited in MIT’s Registry of Standard Biological Parts (including the ones designed by iGEM competition participants themselves) are well characterized, and even if they are, the “parts” may not work together with each other, or with the organism that contains them, as planned.

A January 2010 news feature in Nature, entitled “Five hard truths for synthetic biology”, details the many challenges to the field of synthetic biology. The construction of the “synthetic cell” does little or nothing to help synthetic biologists to meet these challenges.

Our main interest in synthetic biology is in metabolic engineering for use in drug discovery and in production of natural product drugs that are difficult to obtain cheaply from their natural plant sources and are difficult to synthesize chemically. We have two articles on synthetic biology on this blog pervious to the July 12, 2010 article–one dated July 28, 2009 and the other dated September 7, 2009 .

We are much more impressed by some of the work discussed in the September 7, 2009 article than by the creation of the “synthetic cell”. These include, for example, the metabolic engineering studies of Jay Keasling, such as his engineering of yeast and E coli to produce precursors of the antimalarial drug artemisinin. (For example, see this 2006 Nature article.) Dr. Keasling and his colleagues had to contend with several genes that did not work together as envisioned, and to find ways to optimize their expression so that they would work together to produce significant quantities of the desired product. Dr. Keasling says that it took approximately 150 person-years to complete this project. The pharmaceutical company Sanofi-Aventis has been scaling up Dr. Keasling’s artemisinin yeast production system, and plans to sell its product cheaper than artemisinin produced by current methods (i.e., from its natural plant source) by 2012.

We are also impressed by George Church’s automated method for optimizing metabolic engineering pathways, which was the focus of our September 7, 2009 article. This might make the work of metabolic engineers, epitomized by the Keasling groups’s work on artemisinin, faster, easier, and less expensive.

Finally, engineered mycoplasma are unsuitable for making medicines. They are too fragile, and their genomes are too small. For making medicines, one needs a “workhorse” organism that can take the extensive genetic manipulation needed to engineer new metabolic pathways, without losing viability and while maintaining a reasonable growth rate. And cultures of engineered microorganisms must be capable of being scaled up to industrial levels. Metabolic engineers have used such “workhorse” organism as E. coli, Saccharomyces cerevisiae, Streptomyces lividans, and Myxococcus xanthus.

Dr. Venter, through his company Synthetic Genomics, Inc. (SGI, La Jolla, CA) intends to work on synthetic genomics solutions for the production of biofuels. SGI has an alliance with the Exxon Mobil Research and Engineering (EMRE) group to generate biofuels from algae. Dr. Venter claims that SGI will make use of the tools and technologies from the synthetic genome research (together with more traditional metabolic engineering methods) to build “an entire algae genome so we can vary the 50 to 60 different parameters for algae growth to make superproductive organisms.” An alga that would be capable of being scaled up to produce commercial amounts of biofuels would have to be a real “workhorse” organism. And engineering of such algae would be an impressive achievement, beyond the level of a mere technical tour de force as with the “synthetic” mycoplasma cell. We (along with the rest of the world) await further progress on this project.

In late May of 2010–as most of you probably know–J. Craig Venter announced that he and his colleague at the J. Craig Venter Institute (JCVI) (Rockville, MD) had created a “synthetic cell”. This was a bacterial cell containing an entire genome that had been synthesized based on a nucleotide sequence stored on a computer, which had then been inserted into the cytoplasm of another bacterial cell. The JCVI paper was first published online in Science on May 20. It has now appeared in print in the July 2, 2010 issue of Science, where it made the cover of the journal.

Commentaries on the JCVI paper were published in Nature (here and here) and in the New York Times, among other places.

To summarize the JCVI report: the researchers assembled sets of chemically synthesized oligonucleotides, in stages, into a 1.08 million base pair DNA molecule with the slightly modified sequence of the genome of the bacterium Mycoplasma mycoides. Among the slight modifications were “watermarks”, i.e., four added sequences in nonessential genome regions that identify the researchers, and enable researchers to differentiate synthetic genomes from natural ones. The researchers then transferred the synthetic M. mycoides genome into the closely related bacterium M. capricolum, where then new genome took over the cells, resulting in bacteria that expressed the proteome of M. mycoides. The resulting cells were dubbed “synthetic cells”.

The work of the JCVI researchers was a technical tour de force. It required the accurate sequencing of the M. mycoides genome, as well as advanced technologies for accurate chemical synthesis of oligonucleotides, oligonucleotide extraction and assembly, and transplantation into recipient bacterial cells. There were many stumbling blocks in this process. The final stumbling block was a single base pair deletion in an essential gene for chromosomal replication. Once they discovered and corrected this error, the researchers were able to successfully transplant the synthetic genome and get it to commandeer the recipient M. capricolum cells, resulting in expression only of M. mycoides proteins as directed by the synthetic genome.

In evaluating the importance of the “synthetic cell” beyond this technical virtuosity, there are two perspectives–philosophical and scientific/technological. From the philosophical point of view, some commentators hailed this work as the final refutation of vitalism, i.e., the contention that there is something special about processes in living organisms that cannot be artificially created from nonliving systems. Interestingly, the commentators who expressed this conclusion are philosophers and bioethicists. (See, for example, the statements of philosopher Mark Bedau and bioethicist Arthur Caplan in the 27 May Nature discussion article.)

However, as one can see from the same discussion article, scientists–including leading working synthetic biologists–know better. The old biologists’ dictum, “all life comes from life”, espoused by Louis Pasteur among others, still holds. Not only were living M. capricolum cells required for the creation of “synthetic” M. mycoides cells, but the researchers utilized the yeast homologous recombination system in vivo to assemble their “synthetic” genome.

From the scientific and technological perspective, what has the “synthetic cell” project done to advance synthetic biology, beyond the demonstration of technical virtuosity?  The answer of most leading scientific commentators, including working synthetic biologists, is “not very much”.

We agree. We’ll discuss why in Part 2 of this article.

In a previous blog post, we talked about the role of metabolic engineering and synthetic biology in facilitating a return to natural products as drug candidates in drug discovery and development. In the August 13 issue of Nature, George Church (Harvard Medical School) and his colleagues reported on their new method for accelerating the optimization of metabolic pathways to produce medically and industrially useful natural products.

The Church group calls its technology Multiplex Automated Genome Engineering (MAGE). MAGE is an efficient, inexpensive, automated system to simultaneously modify many targeted chromosomal locations (such as genes or regulatory elements) across a large population of cells, through the repeated introduction of synthetic oligonucleotides. A bacteriophage-mediated homologous recombination system is used to replace the targeted sequences with sequences of the introduced oligonucleotides. As the result of this process, researchers obtain a heterogeneous, highly diverse population of cells. Researchers may subject this population to selection for a desirable property, such as more efficient production of a desired product. The selected cells may then be subjected to additional rounds of MAGE, followed by additional rounds of selection. The result is the evolution of strains that efficiently produce the desired product. These strains may be scaled up to produce the product for research or commercial purposes.

The Church group chose to demonstrate their MAGE technology by optimizing a pathway in Escherichia coli for production of the carotenoid lycopene (the red pigment found in tomatoes and watermelons, which is valued as a nutraceutical). These researchers’ approach to utilizing and optimizing this pathway builds upon the work of leading metabolic engineers Jay Keasling (University of California at Berkeley) and Gregory Stephanopoulos (MIT).

Carotenoids such as lycopene are members of a larger class of compounds called isoprenoids. Another class of isoprenoids is the terpenoids. As discussed in our previous blog post, terpenoids include numerous marketed natural product drugs, and this class of compounds is also of interest to researchers interested in discovering novel drugs. Because of common pathways for biosynthesis of precursors of carotenoids and terpenoids, Church’s work on optimizing production of lycopene in E. coli is relevant to researchers interested in applying synthetic biology to the synthesis and study of terpenoid drugs.

The pathway in E. coli (and in other prokaryotes) for synthesis of isoprenoids is known as the DXP (deoxyxylulose-5-phosphate) pathway. This is in contrast to the better-known mevalonate pathway, which is found principally in eukaryotes and in archaea. We discussed Dr. Keasling’s engineering of the mevalonate pathway in yeast and in E. coli (the latter of which was engineered to express this exogenous pathway) to produce terpenoid drugs in our 2007 synthetic biology report. A review of work on metabolic engineering of both the mevalonate pathway and the DXP pathway by the Keasling group and by others can also be found in a 2007 paper by Drs. Withers and Keasling.

In order to utilize the E. coli DXP pathway to produce lycopene, researchers must engineer the bacteria to express the enzymes that catalyze the final steps in lycopene biosynthesis (i.e., the three enzymes that convert the final product of the DXP pathway to lycopene). The Church group transfected their starting E. coli strain with a plasmid containing the genes (derived from another species of bacterium) for these three enzymes. The resulting E. coli strain produced lycopene at a basal level. It was that strain that the researchers subjected to MAGE.

The researchers used the MAGE system to target each of 20 endogenous E. coli genes in the DXP pathway. For each gene, they designed 90-mer oligonucleotides that contained variants of the gene’s ribosome binding site (RBS), in order to replace the endogenous RBS with one that would give more efficient translation of mRNA into protein. They also designed oligonucleotides to knock out four endogenous genes that encode enzymes that siphon off intermediates from the DXP pathway, in order to increase the flux through the DXP pathway to improve lycopene production. The total pool of oligonucleotides was in the hundreds of thousands. The goal was to optimize 24 genes simultaneously in order to achieve maximal lycopene production.

The researchers added the cells and oligonucleotides to the MAGE system, cycling the cells through oligonucleotide delivery (via electroporation), growth, and washing cycles, yielding billions of genetic variants per day. Every 24 hours, the researchers selected the variants that produced the reddest colonies, and thus the most lycopene. After only three days, the procedure yielded variants that exhibited a fivefold greater lycopene production than the starting strain, with a greater yield (approximately 9,000 micrograms per gram dry cell weight) than previously documented.

E. coli strains with an optimized DXP pathway, as developed by the Church group, could in principle be used to produce other isoprenoid compounds, including terpenoid therapeutics. In order to do so, researchers would need to transfect specific sets of genes to carry out the final steps of the biosynthesis of their desired compounds into the strains, instead of the specific lycopene biosynthesis genes used by the Church group. They might also use methods such as the “designed divergent evolution” technology developed by the Keasling group, to develop variants of enzymes that carry out the final steps of the biosynthesis of terpenoids, in order to discover novel terpenoid drugs that are not found in nature.

MAGE, which allows researchers to simultaneously optimize the expression of large sets of genes in a metabolic pathway, contrasts with traditional metabolic engineering, which is typically a slow process in which genetic constructs are introduced into cells one at a time. It thus represents a potential advance. However, as in the above MAGE-based optimization of lycopene production, applications of MAGE to natural product drug discovery and production will build on the work of metabolic engineers who use more conventional methods.

In the 10 July issue of Science, Jesse W.-H. Li and John C. Vederas of the University of Alberta reviewed the current state of natural products-based drug discovery and development, in a report entitled “Drug Discovery and Natural Products: End of an Era or an Endless Frontier?”

As of 1990, some 80% of marketed drugs were either natural products or analogues based on natural products. Two of the major families of natural products that have been of special interest to drug discovery researchers are the polyketides and the terpenoids. Examples of marketed polyketide drugs include the cancer drug doxorubicin, the antibiotic erythromycin, statins including lovastatin (Merck’s Mevocor) and derivatives such as simvastatin (Merck’s Zocor) and atorvastatin (Pfizer’s Lipitor), and the immunosuppressive drug rapamycin. Examples of marketed terpenoid drugs include paclitaxel (Bristol-Myers Squibb’s Taxol) and the cancer drugs vinblastine and vincristine.

During the 1990s and continuing to the present day, small-molecule drug discovery changed to emphasize libraries of synthetic organic compounds, for use in high-throughput screening (HTS). Many companies abandoned the field of natural products altogether. This was driven by pharmaceutical companies’ pursuit of blockbuster drugs, in order to produce the growth in revenues demanded by the companies’ shareholders.

As the fruits of genomics entered the pharmaceutical arena, HTS of synthetic compound libraries against genomics-derived targets became the governing paradigm of small-molecule drug discovery. Nevertheless, natural products and natural product derivatives still accounted for around 50% of newly approved drugs between 2005 and 2007. Over 100 natural products and natural product derivatives are now in clinical studies.

In the 1990s and 2000s, drug discovery researchers emphasized synthetic compounds over natural products because they are easy to synthesize, and are more amenable to use in HTS. They allow researchers to examine large numbers of compounds in a short amount of time. In contrast, natural products have complex structures, are often difficult to synthesize, may be present as small amounts of active compound in complex mixtures in their natural sources, and present a number of other difficulties. Nevertheless, hit rates with synthetic compound libraries are very low, less than 0.001%. For polyketide natural products, for example, hit rates have been about 0.3%.

Given the low output of marketed drugs (let alone blockbusters) emerging from the synthetic compound library-HTS-large scale genomics paradigm, drug discovery researchers may be drawn to look for alternatives that might give higher rates of productivity. We have spoken of biology-driven drug discovery as an alternative to large-scale genomics-driven drug discovery in earlier posts. This deals with the target discovery and validation side of drug discovery. Researchers may also want to look at the chemistry side of small-molecule drug discovery.

The authors of this review suggest that they take a new look at natural products. In their report, they review new technologies for gaining access to, screening, and synthesizing novel natural products and natural product derivatives. There are also a vast number of organisms that are yet to be explored for natural products with potential pharmaceutical activity. The authors of this review therefore believe that the field of natural product medicines may well experience a revival in the near future. The low productivity of the current paradigm of drug discovery may provide an additional impetus for researchers and companies to return to natural products as a source of new small-molecule drugs.

Among the technologies that the authors discuss is an application of synthetic biology known as metabolic engineering. This involves reengineering of natural metabolic pathways in microorganisms to produce useful pharmaceutical products, including difficult-to-synthesize natural products and novel natural product derivatives. Our report on synthetic biology, including metabolic engineering to produce terpenoid and polyketide natural products, was published by Decision Resources in 2007.

An example of a biotech company that is commercializing the fruits of metabolic engineering is Biotica Technology (Cambridge, U.K.). Biotica is using metabolic engineering to discover and develop polyketide natural product derivatives for treatment of such diseases as cancer, hepatitis C, asthma, and inflammation.