January 2008 Archives

Craig Venter and his crew have just published a paper in Science demonstrating synthesis of a complete bacterial chromosome.  Venter let the cat out of the bag late last year in an interview with The Guardian, which I wrote about a few weeks ago, here: "Updated Longest Synthetic DNA Plot".

As a technical achievement, the paper, by Gibson, et al., is actually quite nice.  The authors ordered ~5kB gene cassettes from Blue Heron, DNA 2.0, and GENEART, and then used a parallel method to assemble those cassettes into the ~580kB full genome in just a few steps.  They contrast their method, which may be generalizable to any sequence, with previous research:

All [the previous] methods used sequential stepwise addition of segments to reconstruct a donor genome within a recipient bacterium. The sequential nature of these constructions makes such methods slower than the purely hierarchical scheme that we employed.

The Itaya and Holt groups found that the bacterial recipient strains were unable to tolerate some portions of the donor genome to be cloned, for example ribosomal RNA operons. In contrast, we found that the M. genitalium ribosomal RNA genes could be stably cloned in E. coli BACs. We were able to clone the entire M. genitalium genome, and also to assemble the four quarter genomes in a single step, using yeast as a recipient host. However, we do not yet know how generally useful yeast will be as a recipient for bacterial genome sequences.

The team was evidently unable to successfully use the synthetic chromosome to boot up a new organism.  It turns out that one of the techniques they developed in fact gets in the way of finishing this final step.  There is an interesting note, added in proof, at the end of the paper:

While this paper was in press, we realized that the TARBAC vector in our sMgTARBAC37 clone interrupts the gene for the RNA subunit of RNase P (rnpB). This confirms our speculation that the vector might not be at a suitable site for subsequent transplantation experiments.

So, Gibson, et al., made really interesting technical progress in developing a method to assemble large, (seemingly) arbitrary sequences.  However, their goal of booting up a synthetic chromosome using the assembly technique is presently stymied by one of the technologies they are relying on to propagate the large construct in yeast.  As for the goal of "synthetic life" as defined by constructing a working genome from raw materials, they are close, but not quite there.  Given the many different wasy of manipulating large pieces of DNA within microbes, it won't be long until the Venter Institute team gets there.

Andrew Pollack of the NYT quotes Venter as saying, “What we are doing with the synthetic chromosome is going to be the design process of the future."  This is a bit of a stretch, because no one in their right mind is going to synthesize an entire microbial genome for a real engineering project, with real costs, anytime soon.  Any design process that involves writing whole genomes is going to be WAY in the future.  As I wrote in the "Longest Synthetic DNA" post:

The more interesting numbers are, say, 10-50 genes and 10,00-50,000 bases.  This is the size of a genetic program or circuit that will have interesting economic value for many decades to come.  But while assembling synthetic constructs (plasmids) this size is still not trivial, it is definitely old news.  The question is how will the cost for constructs of this size fall, and when can I have that DNA in days or hours instead of weeks?  And how soon before I can have a desktop box that prints synthetic DNA of this length?  As I have previously noted in this space, there is clear demand for this sort of box, which means that it will happen sooner or later.  Probably sooner.

The Gibson, et al, Science paper doesn't say how many person-hours the project took, nor does it say exactly how much they spent on their synthetic construct (presumably they got a nice volume discount).  The fact that the project isn't actually finished demonstrates that this is hardly a practical engineering challenge that will find a role in the economy anytime soon.

That said, I could certainly be wrong about this assertion, particularly if other technical approaches crop up, as may well happen.  In the NYT story Venter is quoted as saying that, "I will be equally surprised and disappointed if we can’t do it in 2008.”  And they probably will, but what is the real impact of that success? 

The NYT story, by Andrew Pollack, carries the unfortunate title, "Scientists Take New Step Toward Man-Made Life".  Not so much.  Even if Venter and colleagues do get their chromosome working, they will have demonstrated not "man-made" life, but rather a synthetic instruction set running in a pre-existing soup of proteins and metabolites in a pre-existing cell.  It's really no different than getting a synthetic viral genome working in cell culture, which is old news.  Show me a bacterial cell, or something else obviously alive, from an updated Miller-Urey experiment and then I will be really impressed.  Thus the Gibson paper represents a nice technical advance, and a good recipe for doing more science, but not much in the way of a philosophical earthquake.

Without the ability to easily -- very easily -- print genomes and get them into host cells at high efficiency and low cost, building synthetic genomes will remain just interesting science.

The New York Times gets a story title backwards

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The story itself is right on the money, mind you -- I highly recommend reading it -- but the title, "An Oil Quandary: Costly Fuel Means Costly Calories", is bass-ackwards.  That title, probably courtesy of an editor, rather than the reporters, would be accurate for ethanol but has the effect before the cause for vegetable oil-based biodiesel.

Indeed, the story is the same as the one Bio-era has been telling for the last year.  "Chomp! Chomp! Fueling a new agribusiness", written (mostly by Jim Newcomb) for CLSA, nailed all the trends early on; rising income, rising meat consumption, grain use for food and feed, water supply issues, carbon emissions, and government mandates for biofuel use.  It all adds up to a big mess, for the time being.

As I wrote last year while in Hong Kong (See "Asia Biofuels Travelblog, Pt. 2"), after having just been on the ground in Malaysia and Singapore, food use has driven the price of of palm and other vegetable oils well above the wholesale price for finished petrodiesel.  Planting more oil palms, even if done on land that has already been cleared (i.e., not on virgin jungle or on drained peat bogs), is unlikely to ease price pressures because demand is climbing much faster than supply could possibly keep up (see the "Travelblog" post for some rough numbers).  In other words, there is plenty of price pressure to keep cutting down forests and draining peat bogs, carbon emissions be damned.  Prices are probably going to stay high for quite a while.

As the NYT story notes, biodiesel refineries are sitting idle all over the place because the feedstock is way too expensive to turn into fuel.  Far better, and more profitable, to eat it.  The heart of the matter is that, as the Times says, "Huge demand for biofuels has created tension between using land to produce fuel and using it for food."  The arable land is the key issue, and the only way the ongoing collision between food and fuel is going to be resolved is by using non-food feedstock to make fuel, to grow that feedstock on land that cannot be used to produce food at market prices, and produce biofuels using new technologies.  Synthetic biology, various grasses, and sugar from Brazil seem to be the way to go (see my earlier posts "The Need for Fuels Produced Using Synthetic Biology" and "The Intersection of Biofuels and Synthetic Biology").  Hmmm...I still need to post something about switchgrass, miscanthus, and prairies.  Maybe next week.

I'm headed to Houston on Monday for a Roundtable on biofuels run by Bio-era, "Biotech Biofuels & the Future of the Oil Industry".  Companies in the oil industry, agbiotech, and synthetic biology will all be there.  Should be interesting.

High Yield Ethanol Fermentation from Synthesis Gas

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The New York Times is reporting that GM has directly invested in a waste-to-ethanol company in order to help supply biofuels.  Coskata (another Khosla-funded company) has a proprietary combined industrial-biological process for using synthesis gas (CO and H2) to produce ethanol.  Here is the NYT story, by Matthew Wald.

This announcement is interesting to me for several reasons.  First, it turns out I was told all about the Coskata process late last year (though not the GM investment), but I was so busy I didn't tune in sufficiently and so completely missed the significance.  Oops.

Second, in about 2002, I suggested to GM's upper management that they should start thinking of themselves as a "transportation solutions" company rather than just a company that sells cars, and that they invest in providing alternative fuels to ensure that their advanced technology cars would have something to burn. (As the NDA has long since expired, I will connect the dots and point interested readers to an earlier post of mine on producing hydrogen from waste.)  Think W. Edwards Deming and buggy whip manufacturers -- over the next two decades selling cars by themselves is rapidly going to become a losing business model in developed countries as manufacturing practices change and as carbon becomes a bigger issue.  I don't claim that my suggestion five years ago is what got GM started down this road, but I am certainly interested to see that they have made the decision.

The NYT story quotes a number of people commenting on GM's investment, and I think this is the most interesting one, because it is so wrong:

“I don’t really see the logic of it,” said Christopher Flavin, president of the Worldwatch Institute, a Washington environmental group. “It’s not particularly an industry they know well, or have expertise in.” Companies like G.M., he said, could be more effective by concentrating on the fuel efficiency of their products.

GM is now facing enormous pressure to reduce the carbon emissions from its vehicles, in part by increasing fuel efficiencies.  But that isn't the whole story.  Carbon emissions can fall much faster by switching to new fuels, but the extra cost that goes into building engines able to burn those fuels is wasted without access to the fuel.  My earlier suggestion to GM was in the context of using hydrogen as that fuel, but the argument is the same for any other fuel.  Without a sufficient supply of the fuel, why would anyone bother to pay extra for a vehicle that could have lower emissions if only the fuel were available? 

The Coskata website is rather thin on details, but basically they describe a microbe that can convert CO and H2 to ethanol on the fly.  I am absolutely certain the NDA covering the conversation in which I learned about this is still in effect, which limits my ability to say more than what has been published elsewhere.

What I can say is that, if the technology proves to be as efficient and versatile as is claimed, this strategy makes a great deal of sense.  From the NYT story:

If it can be done economically, the Coskata process has three large advantages over corn-based ethanol, according to General Motors. First, it uses a cheaper feedstock that would not compete with food production. Second, the feedstock is available all over the country, a crucial point since ethanol cannot be shipped from the corn belt to areas of high gasoline demand in existing pipelines.

As I have written in this space many times (see, for example, "The Need for Fuels Produced Using Synthetic Biology"), getting away from competition with food is the most important next step in increasing biofuel production.  Diversifying feedstocks to include waste products is critical.

Finally, it is interesting to speculate about the possibility of combining Coskata's synthesis gas eating microbe with the non-fermentative biofuel synthesis I wrote about last week.  Fermentation produces lots of stuff besides ethanol, and ethanol is toxic to most microbes above minimal concentrations.  Besides, ethanol sucks as a biofuel.  So if you could patch the biosynthesis technology that Gevo (another Khosla-funded company, hmmm...) just licensed from UCLA into a bug that eats synthesis gas, you would have a generalized method for taking any organic trash and converting it via synthesis gas into many useful materials, starting with fuels.  Put all together and what do you get?

Say it all together now: "Distributed Biological Manufacturing" (PDF).

A paper in last week's Nature demonstrated a combination of genetic modifications that allowed E. coli to produce isobutanol from glucose at 86% of the theoretical maximum yield.  Please people, slow down!  How am I supposed to finish writing my book if you keep innovating at this rate?

I jest, of course.  Mostly.

Atsumi, et al., exploit non-fermentative synthesis to maximize the production of molecules that could be used as biofuels, while minimizing parasitic side reactions that serve to "distract" their microbial work horse (here is the abstract in Nature).  The authors deleted 7 native genes, added several more from yeast and other microbes, and also added a plasmid containing what looks like another 6 or so genes and regulatory elements.  The plasmid was used to overexpress genes in a native E. coli synthesis pathway.  So call it ~15 total changes.

While the various genetic changes were made using traditional cloning techniques, rather than by synthesis, I would still put this project squarely in the category of synthetic biology.  True, there is no evident quantitative modeling, but it is still a great story.  I am impressed by the flavor of the article, which makes it sound like the project was cooked up by staring at a map of biochemical process (here is a good one at ExPASy -- you can click on the map for expanded views) and saying, "Hmmm... if we rewired this bit over here, and deleted that bit over there, and then brought in another bit from this other bug, then we might have something."  Molecular Legos, in other words.

As far as utility in the economy goes, the general method of engineering a biosynthesis pathway to produce fuels appears has, according to the press release from UCLA, been licensed to Gevo.  Gevo was founded by Francis Arnold, Matthew Peters, and Peter Meinhold of the California Institute of Technology and was originally funded by Vinod Khosla.

It is not clear how much of the new technology can be successfully claimed in a patent.  Dupont a published application from last spring (Update -- typed too fast)  Dupont had an application published last spring that claims bugs engineered to produce fuels via the Ehrlich pathway, and it appears to be very similar to what is in the Atsumi paper described above.  Here is the DuPont application at the USPTO, oddly entitled "Fermentive production of four carbon alcohols".  The "four-carbon" bit might be the out for the UCLA team and Gevo, as they demonstrate ways to build molecules with four and more carbons.  Time, and litigation, will tell who has the better claims.  And then both groups probably have to worry about patents held by Amyris, which is probably also claiming the use of engineered metabolic synthesis for biofuels.  Ah, the joys of highly competitive capitalism.  But, really, it is all good news because all the parties above are trying to move rapidly beyond ethanol.

I am no fan of ethanol as a biofuel, as it has substantially lower energy density than gasoline and soaks up water even better than a sponge.  If ethanol were the only biofuel around, then I suppose we would have to settle for it despite the disadvantages.  But, obviously, new technologies are rapidly being demonstrated that produce other, better, biofuels.  The Atsumi paper serves as yet more evidence that biological technologies will prove a substantial resource in weaning ourselves from fossil fuels (see  my earlier posts "The Need for Fuels Produced Using Synthetic Biology" and "The Intersection of Biofuels and Synthetic Biology").

New method for "bottom-up genome assembly"

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Itaya, et al., have published a new method for assembling ~5kB DNA fragments into genome-sized pieces in this month's Nature Methods (PubMed).  Jason Kelly has launched a blog, Free Genes, where he describes the new method.  Welcome to the blogosphere, Jason.

I won't add anything to Jason's post, other than to note that because Itaya's method exploits a recombination mechanism present in a microbe, there is no need to manipulate large pieces of DNA "by hand".  This is a significant advantage over methods that require lots of pipetting between PCR steps, which exposes the growing DNA to fluid shear.  The reliance upon natural mechanisms for assembly might mean the method is better suited to the garage than something that uses fluid transfer.

Finally, building ~5kB segments doesn't appear to be such a big deal at this point.  While Itaya's method isn't completely general, and as described may be a bit slow, it should be widely useful to anyone who has an in-house method for making gene-sized pieces of DNA and who doesn't want to pay a foundry to assembly even larger pieces.

(Update: Oops.  I forgot to add that this sort of thing is just what I suggested in my previous post, when I observed that while Venter may have made excellent progress in building an artificial chromosome he certainly doesn't have a lock on building new organisms.)

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