High yield biofuels production using engineered "non-fermentative" pathways in microbes.

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"

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.)

Updated "Longest Synthetic DNA" Plot

With the reported completion of a 580 kB piece of DNA by Venter and colleagues, it is time to update another metric of progress in biological technologies.  Assuming the report is true, it provides evidence that the technological ability to assemble large pieces of DNA from the short oligonucleotides produced by DNA synthesizers is keeping up with the productivity enhancements enabled by those synthesizers (see my prior post "Updated, Um, Carlson Curve for DNA Synthesis Productivity").  That said, this is an accomplishment of art and science, not of commerce and engineering.  The methods are esoteric and neither widespread nor sufficiently low cost to become widespread.

The news report itself is a couple of months old now.  It yet to be confirmed by scientific publication of results, so I am breaking my habit of waiting until I can see the details of the paper before including another point on the plot.  Perhaps I just need something to do as a break from writing my book.

In any event, in the 6 October, 2007 edition of The Guardian, Ed Pilkington reported, "I am creating artificial life, declares US gene pioneer":

The Guardian can reveal that a team of 20 top scientists assembled by Mr Venter, led by the Nobel laureate Hamilton Smith, has already constructed a synthetic chromosome, a feat of virtuoso bio-engineering never previously achieved. Using lab-made chemicals, they have painstakingly stitched together a chromosome that is 381 genes long and contains 580,000 base pairs of genetic code.

It does not appear, from Mr. Pilkington's story, that Venter et al have yet inserted this mammoth piece of DNA into a cell.  Though Craig Venter is supposedly "100% confident" they can accomplish this, and as a result will boot up a wholly artificial genome running a semi-artificial organism; "The new life form will depend for its ability to replicate itself and metabolise on the molecular machinery of the cell into which it has been injected, and in that sense it will not be a wholly synthetic life form."

The Guardian story includes a comment from the dependably well-spoken Pat Mooney, director of the ETC Group.  Says Mooney,  "Governments, and society in general, is way behind the ball. This is a wake-up call - what does it mean to create new life forms in a test-tube?"

Here is an open letter to Mr. Mooney:

Dear Pat,

It doesn't mean a damn thing.  Except that it helps you raise more money by scaring more people unnecessarily, so that you can go on to scare yet more people.  Have fun with that.

Best Regards,

Rob Carlson

PS Great business model. 

I just can't get really excited about 580 kB of synthetic DNA.  First, while interesting technically, the result is entirely expected.  People keep saying to me that it is really hard to manipulate large pieces of DNA in the lab, and to this I say many things we do are really hard.  Besides, nature has been manipulating large pieces of DNA very successfully for a while now.  Say, three billion years, give or take.  It was inevitable we would learn how to do it. 

Second, I know of a few individuals who are concerned that, because there is insufficient funding for this sort of work, Venter and his crew will now have some sort of lock on the IP for building new organisms.  But it is so very early in this technological game that putting money on the first demonstrated methodology is just silly.  Someone else, probably many different someones, will soon demonstrate alternatives.  Besides, how many times are we going to need to assemble 580,000 bases and 381 genes from scratch?  The capability isn't really that useful, and I don't see that it will become useful anytime soon.

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. 

Third, the philosophical implications of constructing an artificial genome are overblown, in my humble opinion.   It is interesting to see that it works, to be sure.  But the notion that this demonstrates a blow against vitalism, or against other religious conceptions of life is, for me, just overexcitement.  Venter and crew have managed to chemically synthesize a long polymer, a polymer biologically indistinguishable from naturally occurring DNA; so what?  If that polymer runs a cell the same way natural DNA does, as we already knew that it would, so what?  Over the last several millennia religious doctrine has shown itself to be an extremely flexible meme, accommodating dramatic changes in human understanding of natural phenomena.  The earth is flat!  Oh, wait, no problem.  The earth is at the center of the universe!  No?  Okay, we can deal with that.  Evolution is just another Theory!  Bacteria evolve to escape antibiotics?  Okay, God's will.  No problem. I can't imagine it will be any different this time around.

Finally, it is worth asking what, if any, implications there are for the regulatory environment.  The Guardian suggests, "Mr Venter believes designer genomes have enormous positive potential if properly regulated."  This is interesting, especially given Venter's comments last winter at the initial public discussion of "Synthetic Genomics: Options for Governance".  I don't know if his comments are on record anywhere, or whether my own public comments are for that matter, but Venter basically said "Good luck with regulation," and "Fear is no basis for public policy."  In this context, I think it is interesting that Venter is not among the authors of the report.

I just finished writing my own response to "Options for Governance" for my book.  I can't say I am enthusiastic about the authors' conclusions.  The  authors purport to only present "options".  But because they examine only additional regulation, and do not examine the the policy or economic implications of maintaining the status quo, they in effect recommend regulation.  One of the authors responded to my concerns of the implicit recommendation of regulation with, "This was an oversight."  Pretty damn big oversight.

Today's news provides yet another example of the futility of regulating technologies to putatively improve security.  Despite all the economic sanctions against Iran, despite export restrictions on computer hardware, scientists and engineers in Iran report that they have constructed a modest supercomputer using electronic components sold by AMD.  Here is the story at ITNews (originally via Slashdot).  Okay, so the Iranians only have the ability to run relatively simple weather forecasting software, and it may (may!) be true that export restrictions have kept them from assembling more sophisticated, faster supercomputers. (I have to ask at this point, why would they bother?  They are rolling in dollars.  Why not just pay somebody who has a faster machine to do the weather forecasting for you?  It suggests to me that they have pulled the curtain not from their best machine, but rather from one used to be used for weapons design and is now gathering dust because they have already built a faster one.)  Extending this security model to biological technologies will be even less successful.

Export restrictions for biological components are already completely full of holes, as anyone who has applied for an account at a company selling reagents will know.  Step 1: Get a business license.  Step 2: Apply for account.  Step 3: Receive reagents in mail.  (If you are in a hurry, skip Step 1; there is always someone who doesn't bother to ask for it anyway.)  This particular security measure is just laughable, and all the more so because any attempt to really enforce the legal restrictions on reselling or shipping reagents would involve intrusive and insanely expensive physical measures that would also completely crimp legitimate domestic sales.  I can only imagine that the Iranians exploited a similar loophole to get their AMD processors, and whatever other hardware they needed.

Well, enough of that.  I have one more chapter to write before I send the book off to reviewers.  Best get to it.

Updated, um, Carlson Curve for DNA Synthesis Productivity

It seems that productivity improvements in DNA synthesis have resumed their previous pace.  As I noted in Bio-era's Genome Synthesis and Design Futures, starting in about 2002 there was a pause in productivity improvements enabled by commercially available instruments.

According to the specs and the company reps I met at iGEM 2007, a single Febit "Geniom" synthesizer can crank out about 500,000 bases a day and requires about 30 minutes of labor per run.  It looked to me like the number should be closer to 250KB per instrument per day, so I made an executive decision and allowed that the 16 synthesizers one person could run in a day could produce 2.5 megabases of single-stranded ~40-mers per day.  This in part because there is some question about the quality of the sequences produced by the particular chemistry used in the instrument.  It was asserted by the company reps that the Geniom instruments are being adopted by major gene synthesis companies as their primary source of oligos.  Note that running all those instruments would cost you up front just under US$ 5 million, without volume discounts, for 16 of the $300,000 instruments (plus some amount for infrastructure).

The quality of the DNA becomes particularly important if you are using the single-stranded oligos produced by the synthesizer to assemble a gene length construct.  To reiterate the point, the 2.5 megabases per day consists of short, single-stranded pieces.  The cost -- labor, time, and monetary -- of assembling genes is another matter entirely.  These costs are not really possible to estimate based on publicly available information, as this sort of thing is treated as secret by firms in the synthesis business.  Given that finished genes cost about 10 times as much as oligos, and that synthesis firms are probably making a decent margin on their product, the assembly process might run 5 to 8 times the cost of the oligos, but that is totally a guess.  (Here is a link to a ZIP file containing some of the graphics from the Bio-era report, including cost curves for gene and oligo synthesis.)

One final note: the Febit reps suggested they are selling instruments in part based on IP concerns of customers.  That is, a number of their customers are sufficiently concerned about releasing designs for expression chips and oligo sets -- even to contract manufacturers under confidentiality agreements -- that they are forking over $300,000 per instrument to maintain their IP security.  This is something I predicted in Genome Synthesis and Design Futures, though frankly I am surprised it is already happening.  Now we just have to wait for the first gene synthesis machine to show up on the market.  That will really change things. 

How big is the Bio-economy?

The words "biotechnology" and "biotech" are often used by the press and industry observers in limited and inconsistent ways.  Those words may be used to describe only pharmaceutical products, or in another context only the industry surrounding genetically modified plants, while in yet another context a combination of biofuels, plastics, chemicals, and plant extracts.  The total economic value of biotechnology companies is therefore difficult to assess, and it is challenging to disentangle the component of revenue due each to public and private firms.

I've managed to get a rough idea of where the money is for industrial biotech, agbiotech, and biopharmeceuticals.  Based on surveys from Nature Biotechnology, the U.S. Government, various organizations in Europe, and several private consulting firms, it appears estimates of total revenues range from US$ 80 to 150 billion annually, where the specific dollar value depends strongly on which set of products are included.  The various surveys that provide this information differ not only in their classification of companies, but also in methodology, which in the case of data summarized by private consulting firms is not always available for scrutiny.  For whatever reason, these firms tend to produce the highest estimates of total revenues.  Further complicating the situation is that results from private biotech companies are self-reported and there are no publicly available documents that can be used for independent verification.  One estimate from Nature Biotechnology, based on data from 2004 (explicitly excluding agricultural, industrial, and environmental biotech firms), suggested approximately 85% of all biotech companies are private, accounting for a bit less than 50% of employment in the sector  and 27% of revenues.

A rough summary follows:  As of 2006, biotech drugs accounted for about US$ 65 billion in sales worldwide, with about 85% of that in the U.S.  Genetically modified crops accounted for another US$ 6 billion, with industrial applications (including fuels, chemicals, materials, reagents, and services) contributing US$ 50-80 billion, depending on who is counting and how.  Annual growth rates over the last decade appear to be 15-20% for medical and industrial applications, and 10% for agricultural applications.

I am not going to go through all the details here at this time.  But the final amount is pretty interesting.  After sifting through many different sets of numbers, I estimate that revenues within the US are presently about US$125 billion, or approximately 1% of US GDP, and growing at a rate of 15-20% annually.

1% of GDP may not seem very large, but a few years ago it was only 0.5%.  At some point this torrid growth will have to slow down, but it isn't clear that this will be anytime soon.  Nor is it clear how large a fraction of GDP that biotech could ultimately be.  That is my next project.

Off to iGEM 2007

I am headed out the door to the 2007 International Genetically Engineered Machines (iGEM) Competition at MIT.  There look to be ~56 teams composed of ~400 students from around the world.  As I am a judge this year, I won't be blogging any more about it until it's over.

I have been looking forward to this for months -- it should be great fun.

When microbes innovate faster than humans do

CNN is reporting that  methicillin-resistant Staphylococcus aureus (MRSA) is afflicting a number of high school students in the U.S.  One student has died from an infection apparently contracted at school, while another 15 or so students in two states have tested positive.

This is getting press in part because of a report out in JAMA that the rate of infection from MRSA around the U.S. could be twice as high as previously thought, with a mortality rate of almost 20%.  (Here is the paper on PubMed: "Invasive methicillin-resistant Staphylococcus aureus infections in the United States".)  MRSA was first observed in the U.S. only in 1981.  Thus over only about 25 years we have produced a bug, through profligate use of antibiotics and poor sanitation, that may be a bigger killer than even HIV.

This while NIH funding has more than doubled, where most of that money has gone to established investigators (See my post, "The Death of Innovation, or How the NIH is Undermining Its Future") doing whatever it is they do that doesn't result in new antibiotics.  Where is the Health in NIH?

I heard yesterday via the grapevine that an NIH review panel failed to award any of 19 worthy new grants to younger investigators because all the money in the program is sopped up by existing grants.  You could argue that we should just increase the NIH budget, to which I would be sympathetic, but it is by no means clear that the present funding is well-spent.

On the use of the word "Biobrick"

A couple of months ago, Drew Endy admonished me via email for using "Biobricks" as a noun.  The trademark, as held by the Biobricks Foundation (BBF), describes a brand, or marque.  The word "Biobricks" is an adjective describing a particular set of things conforming to a particular standard.

I finally had a chance to catch up via phone Drew yesterday, and he clarified why this is important.  All the groups contributing to the MIT Registry of Standard Biological Parts, mainly via the International Genetically Engineered Machines Competition (iGEM), are working hard to make sure all those parts conform to set of rules for physical and functional assembly.  That means, amongst many other requirements, that the ends of the genes have appropriate sequences for manipulation and are sequence optimized for the assembly protocols.  For example, all the EcoR1 restriction enzyme sites need to be at the ends of the part and not in the middle.

It turns out that Drew is seeing lots of "parts" show up in papers and talks, described as "biobricks", that won't be compatible with the growing list of parts in the Registry refrigerators.  Thus the need for a differentiable marque.  From the BBF FAQ:  "The BBF maintains the "biobrick(s)" trademarks in order to enable and defend the set of BioBrick™ standard biological parts as an open and free-to-use collection of standard biological parts."  Thus it seems the BBF will both assert a standard and curate and license a library of parts.

There will be a BBF open workshop 4-6 November at MIT to define technical and legal standards for Biobricks Biobrick parts (it's just awkward, no?), following iGEM 2007 on 2-4 November at MIT.

Which gets me to wondering what other examples there might be of standards being defined and maintained by a foundation, protected with a trademark.  As far as I know, "transistor-transistor logic" (TTL) became a standard simply because Texas Instruments put a bunch of products out and everybody else jumped on board (see Wikipedia).  But nobody protected the marque "TTL", and no one organization curated and licensed a library of TTL parts.  Similarly, if I have got this right, the IEEE discusses and approves standards for hardware and software that manufacturers and programmers can use, but the IEEE does not itself play a role in building or licensing anything.  (Comments?  Randy?  TK?)

So I wonder if the BBF isn't heading out into some unknown territory.  Obviously, the idea of Biobricks Biobrick parts (Argh!) is itself new and interesting, but I wonder what the effect on innovation will be under an apparently new kind of IP regime if one organization is in a position to "defend" not just a a standard but also parts that conform to the standard.  What happens if the leadership (or control) of the BBF changes and suddenly the "open and free-to-use collection" becomes not so open?  And am I free to build/identify a new part as a Biobrick part (!) without submitting it to the Registry or the BBF?  Can I even advertise something as being compatible with the standard on my own, or do I have to have permission from the BBF to even suggest in public that I have something other people might want to use/buy that works with all the other Biobrick™ parts?  And who exactly controls the Registry?  (The "About the Registry" page doesn't appear to answer this question, even though I believe I have heard Drew and Randy Rettburg say in the past that MIT presently controls the IP.  There was also, I believe, some question as to whether some parts in the Registry are actually owned by other organizations.)

So many questions.  It is clear that there is lot's of work to do...

Metabolic rate determines mutation rate

ScienceDaily has a story describing a new paper showing that the rate of protein evolution is subject to allometric scaling.  Actually, now that I have written that, I remember that allometric scaling describes a specfic mathematical relationship between metabolism and body mass, but the paper in question doesn't appear to be online yet so I can't say for sure allometric scaling is the appropriate mechanism to cite.

At any rate, ScienceDaily reports that James Gillooly, and colleagues have shown that: "...A 10-degree increase in temperature across species leads to about a 300 percent increase in the evolutionary rate of proteins, while a tenfold decrease in body size leads to about a 200 percent increase in evolutionary rates."

"Generally, there are two schools of thought about what affects evolution," said Andrew P. Allen, Ph.D., a researcher with the National Center for Ecological Analysis and Synthesis in Santa Barbara, Calif. "One says the environment dictates changes that occur in the genome and phenotype of a species, and the other says the DNA mutation rate drives these changes. Our findings suggest physiological processes that drive mutation rates are important."

That is pretty interesting.  Warm, small animals evidently experience a greater rate of protein evolution than to large, cold ones.  This suggests to me that warm-blooded, smaller animals have an evolutionary advantage because they are better able to produce physiological variation in the context of a changing environment, and thus better able to compete at the species level in the face of natural selection.  The ScienceDaily story doesn't make that point, but I would assume the paper in Biology Letters, when it is published, will.

Here is the press release from the University of Florida.

HTML Version of "Biological Technology in 2050"

The HTML version of my 2000 essay from the Shell/Economist Writing Competition seems to have fallen off the web.  Here is another copy: "Biological Technology in 2050".