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 had been commercializing the fruits of metabolic engineering is Biotica Technology (Cambridge, U.K.). Biotica had been using metabolic engineering to discover and develop polyketide natural product derivatives for treatment of such diseases as cancer, hepatitis C, asthma, and inflammation. (As of March 2013, NeuroVive (Lund, Sweden) acquired a portfolio of polyketide cyclophilin inhibitors from Biotica, which has gone out of business as of January 2013.)