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r I'd'iike to do a littie bit of•sunmarizing and reviewing by virtue of my position on the program. I'd also like to take this opportunity to re- direct the attention of an audience whose principal focus in the past has been on chemistry and chemical processes. My theme is simple: For us to regard chemistry as one universe and biology as another is a luxury which we can no longer afford# furthermore, it really isn't justified at all. Now, we all know that fermentation is big business - that isn't newst and we all know that fermentation products such as antibiotics, beer, cheese '• and the like have total world-wide sales in the tens of billions of dol- lars.. There are many statistics to back this up, and other people have •.mentioned those statistics, so there's no point in boring you by repeating them. . : , ' So, why are wd here7~v Why do I come from Berkeley to say that there's ~r a revolution starting and ve want everybody to know about it and to parti- : cipate in it? Maybe I should agree with John Littlehailes and say, "Thank you very much, but don't bother to participate in it. We'll do okay with-. out you." That, of course, is not why we're here. I think the paradox, ~ the reason this program was arranged, is the fact that to a large extent, people who have not been involved in biology and, frankly, people who have been involved in biology as well, have been inclined to deal with biology, particularly at the industrial level, as some kind of witchcraft. I can think of a specific example. Recently, I spoke to a top microbiologist of ; one of the world's largest beer manufacturers. He told me that although the people in his company who make yeast for baking were grudgingly willing : to consult with his scientific group to find out what the latest develop- ments were, the people working with Brewer's yeast wouldn't even talk to him. As far as they were concerned, brewing was an art and not a science. Here, in this mee_tingy•;we've been reviewing what has been done over the last twenty years to explain, explore and finally decipher the secrets of life. These represent opportunities, if you like, that billions of years of selection and evolution are now handing us on a silver platter. we're being told that up to recently we were only able to observe what Nature did and now we have the ability to influence, to manipulate what Nature can be taught to do. Thus, we are presented with an opportunity.. ' Let me present a useful analogy.. If you think back over twenty-five years to the birth of solid state devices, it is clear that the amazing things now in extensive use, made possible by that breakthrough, really couldn't be predicted at that time.. And I don't think that's any reflection on the appreciation of the magnitude of the breakthrough or the vision at that there was an inability to make accurate predictions about" Clearly time . , just exactly where transistors wouia leao. eeopie aacx in i"U eia not .anticipate digital watches, hana calcuiators ror Sy.yto, Tv games, or tne fact that in a small airplane today there's more sophistication in elec- tronics than'there was in a Boeing 707 when it first went into commercial service. .The lack of precision in vision in 1950 d~d not cloud the fact '- that there was a revolution in the making. Yet'!t is interesting to note'- :- that the caspanies that have been most instrumental, that have ridden the wave of that revolution, have largely been new companies. With very few exeeptions, they were not the companies that one would have expected, in " ; 1950, to take advantage of that ver obvious revolutionl And so we think in biology that here we are again at the very beginnings of a very signifi- 1' ~ y J lU I • '~4'c~ ~

cant'explosion of capabilities and we think thal.the people.•who have the expertise to exploit it should logically take that oppoitunity., It doesn't necessarily follow that tTiey will. I think the last two days represent our attempt to proselytize, to convince those people who know chemical reac- I tions - who know polymers, to recognize just what the opportunity consists' ~ of and to join in, so to speak. For you, it's a natural. ~ i;- ` a^pn~ Herere two immediate examples that show that thingsare hapeni r fast In the laat several years we have witnesaed an lncredible penetra ~^' .- tion ot the world sucrose market by high fructose corn syrup facilitated ' ," by developments in enzymology. Now, this may be something which an enzymoi-" ogist in 1955 could have imagined. iHut its real significance is its enor- mous mous commercial impact. Would a market forecaster in 1955 have believed U "~ the enzymologist or dismissed him as a? N th , dreamerow,e enz ysioloqiet of 1955 is analogous to the evangelical molecular biologist today. Considez :' also insulin, and the recent demonstration at the University of California~;, at San Francisco of the ability to get the gene for rat insulin into a au ' bacterium. The need for insulin is great. We're talking of new markets for new products, in billions of dollars.;. So, it's very real opportunities that we're talking about and I'm .+ going to be saying repeatedly that we're talking about today's technology; today's state of the art. We're not relying on some breakthroughs without which all of this in just talk. Some of the biologists with whom we deal closely eay, "Just tell us what you want to make, and we'll make it." It's a bit of an exaggeration, but the exaggeration is worthwhile if it gets ; your attention. The capability - what can now be accomplished - is really incredible, and the gap between the capability and where things are in industry is equally incredible. I've said the following before in other ,.. forums and I'm sure I lose some potential friends by saying it. Again, allowing in advance that it's a bit of an exaggeration, those of you from' ` the chemical industry will appreciate it. If you go into a large chemical company in this country or abroad and compare the level of sophistication : in its pure and applied research laboratories with what's going on in universities in those same disciplines, you'll be struck by the similari- ties ties rather than by the contrasts. Frequently, you'll see greater sophis-.;, tication in industry. On the other hand, consider biology. Here in a ,q field in which perhaps twenty or thirty Nobel prizes have been awarded inr)s the past twenty years, for powerful discoveries. So powerful, in fact, ,. that a generation ago some people stated that this knowledge was beyond .~.-; Nan's reach. After all, these were the secrets of life itself. Go into4 those biology-based companies, responsible for tens of billions of dollars worth of business based on exploitation, if you like, of the secrets of life. Again, apologizing for the exaggeration, I ass_ert that Louis Pasteura would not have too much trouble understanding what's going on in some of those research labs. There is a tremendous gap between what's going on , there and in the universities. These "secret of life" developments simply have not been exploited. We1__1_, they soon will be -,and that's what,this conference is all about. Now, let me outline how I want to arrange my-remarks.~ riret,'and i. X because I am the final speaker and have been asked to do so,,I'l1 try to~.`.` race very quickly through a general summary of the expositions which „ -° you've already heard. That will be intentionally superlicial.' Also,'I2'll take the opportunity to say a few things about Cetual I'll say'them by way '. of illustration. When I say "we can do this," or "we can do that," I". ~ assume that other people will follow our example - that•s the prica'wa`pay for freedom. The second thing I want to talk about are challenges - chal-- ~? lenges to biologists and challenges to people in the chemical process and i related industries. I think those challengea heve to be met by both ~1 because, as someone said yestezday, half a cycle is no cycle. And finally. lf time permits, I have a few philosophical observations on politics, blicity to which we've all been`c" regulations and the recombinant DNA pu exposed and which has occupied a fair amount of our time for the past year.. .-. . . y ~., .., .. I think biG2e*ists on the one han~tand the ohemiets on Lhe oiher have-w occupied two_diffesent,worlds in the paet., Nevertheless, we all make, eertain common assumptions, as has been explained by the other speakers. We all seem to agree that there are certain problems especially well-suited to biological.solutions.- Generally,.we agree that it'e nice that biologi- l cal reactions are carried out in a coupled manner under mild conditions of . temperature and preasure., ~ ; A I think it's also agreedthat biologicalsysta ailitd~, •,emre ncey,.sue to: _ the manufacture of complex molecules._;,Some of these biological processes : have been expensive and have been deemed attractive for the manufacture of- expenaive compounda. Antibiotica, of course, come to mind.; The current estimetee;as to the annual world market for antibiotics,fall in the broad ~jwrange of 5-10 billion dollars.,. There's a lot of business there. It's a~~ demonstration of great versatility for a chemist to imitate what a cell " does by manufacturing these complex structures from.scratch Stilli=~ ,,.prn cipally, in terms of economics of production, it's the biological system ~tl~ which we all intuitively assume in going to be thewayto go t' ._., ... .M. , „ fiY•. We also take for granted that either in cellsb „il , or•.y!enzymes-you V want a stereoapecific reaction, you think.about biology. cSteroid arodifica~; ? , tions are a specific example . .#,. Now there are a few other things,,perhapsthatwtay-noL b':bi ~:~: ,,~:e soovous but biology in also very nicely suited to address certain industrial ,• situations. Let's assume that you have a mixed reactant stream - either,ll,r; say, a substrate that's mixed with some relatively inert by-product that comes out in the same stream, or, perhaps, with other reactive products. . A biological system, because of the great specificity of enzymes, can be very selective. The enzyme will act only on one of the stream components. Consequently, a most unsuitable substrate stream for certain chemical reactions may pose no problem and, in fact, be very neatly suited to biological reactions. And, by the same token, you can think of piggy- backing different reaction starters in the reactant stream because of the way in which the specific enzymes can go and "pick out" specific targets, Of course, you must design your process properly. Another cliche, but a true one, in that biology opens up the whole' field of renewable resources. In a sentence, biology gives us a way of.jG, •harnessing the sun's energy and channeling it one way or another. We're I; all interested in that opportunity because of the many shortages that are' developing in the supplies of chemicals, as well as of energy itself.;, It's often been mentioned in connection with the so-called "new bio-,'. logy" that there are hurdles, and~who knows how long down the road it's going to be before these hurdles are in fact hurdled. I think it's very;,,, important to stress that very much like the semiconductor/computer indus- , try, the hurdles are being hurdled faster than even the most optimistic people in the field predict from year to year. I can think back and use an illustration from our own history. As many of you know, Dr. Joshua Leder- berg won the Nobel prize for the discovery of sex in bacteria. He's been one of the greatest proponents of this new biology. When Cetus was first formed in 1972 and we were trying to convince some people that here was an :% I recall several ncredible opportunity, occasione when Dr. Lederberg made; „predictions about what we now know as recombinant DNA. Ne, an incurable __ optlmist, described it.ae an astonishing development tnat might happen, ~ reie tat dohe' sometime within our lifetime. Little did healzhwn t hallDr. ; Stanley Cohen, now also a was,going to make this breakthrough in a matter of months. Today agn,jthey-talk abt"nurdles Mhen'they.talk abouti""expressinq" '`~:c:tF~ ... ..... . . ... . . . , . . .. • a gene, which is moved into a bacterial ituation perhaps from a non ~ bacterial situation. Dr. Glaser told us that there are tWo kinds of cells - the simple ones (prokaryotes) and the caeplex dnes (eukaryotes)L;" ~'-Most of the interesting genes are aukaryotic (insulin, antibodies).. Most? i,'~~-'of the easiest and most obvious production organisms are prokaryotes: Until recently it was thought impossible to get a eukaryotic gene to funo- ~ v K3 tion in a prokaryotic host. Xell, that's a hurdle that's been hurdled. 9~ ,.

And, in both directions. Yeast gened have been expressed in E:'coli and ' .~ certain bacterial genes have been expressed in higher plants. So I think the take-home lesson is that things are going very quickly. 2t'a partly a p~^< matter of getting on the bandwagon before it's too late. Certainly there is no excuse for saying you have to wait five to ten years and see what happens. It's happening right now. The technology is right now. It's a`y question of identifying the business opportunities and moving. 'Z;. I've heard it said that Alfred Nobel's will made no arrangements for: . prizes for witchcraft. In other words, all those Nobel prizes I mentioned earlier were made for very real scientific breakthroughs. You've heard refer_ence to most of them. In review, I'll rush through the overall story- very quickly, using, if you like, analogies in polymer chemistry and in ' tape recording. It will take two or three minutes just to get the wordsF out as a basis for things I'd like to say a little later on. - The blueprint for what is specified in biological systems is a polymer called DNA. A key characteristic discovered about DNA is its ability to replicate itself and to keep those instructions sacred from generation to generation. Of course, we already knew that there had to be a blueprint somewhere or else organisms wouldn't breed true, and we know that organisms do breed true. The blueprints are faithfully duplicated. Then, to pursue the blueprint analogy, there's the concept of the working drawing that goes -` out into the field and is referred to when actually constructing things. The working drawing is messenger RNA, another polymer very familiar to you. It's a string of inert chemical moieties, a string of atoms. There's nothing magic about it. Then, we heard the controls describedt we heard about feedback inhibition, and about the small molecules that fall off of the beginning of transcription'signals so that the working drawings are in fact transcribed. These things are all familiar to you, conceptually, and that's the way living systems are organized. Just like any chemical plant, you might say. + + These polymers are proteinst Don Glaser talked about proteins as being three-dimensional pretzels. The shape determines the function. Going back to DNA, we recall that the code in DNA contains four letters, and the words in the language are three-letter words. I guees there's some temptation with all these sexual references to ask why they aren't four letter wordsl ~ But, no, they're three-letter words and they specify the sequence of amino acids that ultimately determine the shape of a protein. Since most pro- teine, are catalysts (enzymes), we intuitively accept that their spacial characteristics determine their specificity. Then we talked about how yoa change the blueprint, the idea being that by changing the enzyme's behav- ior, you ultimately change the behavior of the organism. Then, you have ' mutation, which is juar, changing the coded message, maybe as simply as by changing only one letter in the message. Then we talked about sexual . mating which was called conjugation. We heard about the uptake of naked . ~ DNA, called transformation. Dr. Ingle described an intriguing case where " f the yield of a particular enzyme was increased rather substantially, simply . ?y'by the uptake of DNA from a strain which has that higher capability. Then aj . we have virus infection. We saw those pictures called tranaduction.. Then we have fusion, which is just bringing two cells together - forcinq them sort of to form one cell and get all that DNA together. And, of course, we have the recombinant DNA techniques which Dr. Littlehailes described. '' These are all different ways of changing the coded message ultimately by * yP.} changing the instructions at the blueprint level. , , .• _;. . Why is recombinant DNA technology so revolutionary?,TrI,think here we should go to the metaphor of data stored in a computer on recording tape... The point has been made that we now know the location of many genes,to the extent that one speaker mentioned that, as a matter of fact, we have gene r' banks. We can actually isolate the pieces of DNA that,specify certain functions and put these pieces in bottles. In these terms, old-fashioned mutation is one extreme where the process„ to continue the computer anal- t ogy, is like taking a machine gun and shooting at a computer, hoping that from time to time this will improve the situation by changing a bit of '\ lo i. 17si~6~i~OnQ~. stored data ~.i think yoy-ll'aqrea that that will be a very`rara and randosi r, event. And,iin fact, favorabl L~ ~e mutations are very, very rare events. On the other hand,'recombinant DNA's elegance, the revolution, lies in the , k fact that we now know where the information is stored on those tapes.' Nore ~~~important we now kn h t i .,owowo gon and splice out that information, store `it in a bottlet t in b until we wano uset, ad then comeack and splice it iinto a cell where we do want to have it expressed. ' The change over the last twenty years is truly incrediblea`.pecific qomparison from the real world may help illustrate the point. When peni-_ cillin was first discovered, activity of the producing organism was so low that it was not commercially useful. The productivity of the organism has been slowly incxeaed ttil ll o so praccaevesver the past forty years. How? Almost exclusively by uin thtil "hi sge parcuarmacne gun approach" called mutation. Today, by way of contrast - as Dr. Littlehailes mentioned - one,-_ of our colleagues at Cetus,-in one year, by using these new recombinant DNA techniques has increased the yield of a particular protein far more than was accompliehed in that:forty yr hityfiilli easor o pencn. where, in the natural state, thisicell has three molecules of this particular protein as_ _ a normal component,,now approximately seventy percent of all protein made , by that cell is that one specific molecule. That's incredible amplifiea-.f _ tion., The point I want to stress is that it was done in one year because it waa-done witheyes open intelligently not by random ati Ai ,,,con.,qan,- I'm repeating myself,.but.I want to reiterate that.we're talking.about the state of the art today. : ,; ; Noa, just a little bit_about-Cetus. I,eaid that if:you ehoot bullets at:a computer, the chance that somehow this is going to improve the situa- tion with respect to something you want is very low. ; This ia true using mutation in biological systems. Still,,in some-situations, you can experi- mentally force a population of organisms to move in the direction you want. You can apply selective preseure.. One could call this "forced evolution." If you want, for example, to begin with an organism that cannot synthesize vitamin B and develop from it some mutants that can synthesize vitamin B, the experiment you set up is obvious. You simply withdraw the vitamin B11 mutate, and see who lives. That mutant stands up, waves his arms and sho_uts, "Here I am, I somehow survivedl" This is_ called "selection." There's a further difficulty. It's obvious that we're not trying to move these bugs to do something differently to make them happy, we're :., trying to coerce them to make something now that makes us happy. Conse- quently, in nearly every case that I know of, the desired bugs, besides - being very rare, do not identify themselves by selection. They don't wave their arms. Instoad you have to isolate or "clone" each mutant and_ ask him if he does what you whnt.: If you're clever, he'll tell you. This process is called_ screening, and it's very laborious. we formed our company to try to speed up this screening process. We got our inspiration from Don Gla- ser. As you know he received his Nobel prize in subatomic physics for developing the bubble chamber, which was a way of catching rare evente. _ _ When Don turned his attention to biology,-he once again became interested in the identification, of rare events. He,told you all about that yester- day. That, in fact, was.how we got our starts large scale, computer- ,_.. assisted screening,:,~ ~ ,L - . , v ; . - , ~ . ,, .. ; But,,as they say in:the:computer industry,;•"Garbage In-Garbage Out." Numbers alone can really be very misleading. You can find yourself pro-. cessing a lot of data and be no smarter when you finish. This is why it's so important,;and has been stressed throughout this meeting, that what is being learned.in genetics should be integrated into our screening strate- gies.l Thisiincludes everything from Josh Lederberg's teaching that bac-,„ teriaienjoy`.sex or at least have sex (we don't know how much they enjoy it) to Stan Cohen's,using chemistry, just straight chemistry, enzym& chemistry if you like, to rearrange the genes to perform specific functions. That's my brief commerciall ; There pre a few other points thatsfell between the cracks during, yesterday's.,and today's presentations.. I'd like to mention them to round

out where biology is today because they have some bearing on what it is y4 that we can now accomplish.eugs are very adaptable; we've seen examples of this. ' I mentioned "04 forced evolution. You can select for certain things. I think it's good to'% mention that that means that even if the capability hasn't come to our attention in nature there are ways of forcing bugs to do things which ?: it ' perhaps they nevbr did before. You've heard, perhaps, of the pink mold °)x`LI that lives on rubber. It's not clear that rubber was around during the t s' " organism's evolution, but those capabilities somehow developed. It's P important to keep this in mind so that when we think of a very specific ~' reaction that we want to catalyze, and use a biological system to make the~'x- enzyme to catalyze it, we don't throw up our hands and say. "Well, that's a reaction which never occurred in nature, so it'g unrealistic to expect ..; . biology to have any solution at all." okay. What can we do? When I say "we," I don't mean.only Cetua.` I mean what can anybody do who decides to devote the resources, the attention,~# and the commitment to exploiting biology. I think we can categorize and ey that, first, we can now manufacture products which were previously uneconomical. $econdly, we can now manufacture products which were previ ously impossible. And, thirdly, and most remarkable of all, we can now manufacture products that were previously inconceivable. N Let me deal with each of these in turn. First, we have to re-examine .•' old cliches about the economics of fermentation. When I said I'm from Berkeley, and we're going to talk about revolution, let me remind you of Bob Dylan's song, "The Times They Are A-changing." Ethylene, which used to : coat two to three cents a pound back during World War 11, now costs 12.5 cents a pound in the U.S. and nineteen cents a pound in Brazil. One can now consider producing it from fermentation alcohol. So where biology obviously didn't make it economically in the past, it may win out in the future. There's no need to say any more about that. New capabilities, new pricing structures, new markets and raw material availabilities throughout the world, must all be re-examined. I think that biology is going to find some opportunities which represent a rain check being cashed in, if you like, because of new circumstances. Single cell protein is another speci- fic example. Al Laskin mentioned the fact that agricultural crops are a very important factor in protein considerations. Soybeans may not always_ mewhere in the` be cheap - single cell protein's day will come, at least so world. A second category I mentioned is the manufacture of products pre ~ that imagerp viously impossible. Recall the chimera. Stan Cohen utilizes to describe situations where the genes of different species are contained '..:_ in one living cell, whether by recombinant DNA techniques, or by fusion or by transformation techniques, or by any of the other tech- teehniques,niques. The chimera, in mythology, was a monster with the body of a goat,13,, the head of a lion, and the tail of a dragon. The statement has been made ..- ' that with today's techniques, with cells, we could produce a real chimera, if it weren't for the fact that dragon cells are a little bit hard to come by. And recombinant DNA is not our only tool. Dr. Nickell talked about plant cell culture. Analogous human and other animal cell cultures can produce human proteins. It may turn out, with some special human products such as interferon or antibodies, that human cell culture may be the pre- ferable way to make them. The point ie that these various techniques are available at once today and they should all be reexamined.' Let's first talk about human proteins such as insulin. Human insulin is a better i` product for a diabetic than is either pig or cow insulin. But you get pig insulin or cow insulin from a slaughter house and, until very recently, there was no demonstration of how you could produce human insulin. Well, recent developments at the University of California at San Francisco point:• the way. Undoubtedly, some time within the next few yeare, we will see the t's a product that wae pss- baeterial produetion of human insulin. Tha viously impoaeible. Another useful human protein family - the antibio •' tics - may represent the means used ultimately to fight all infectious w~ .-. ssWszzoooi ~ .~.~lz* . ~.. . '~.'..• 4. ' J... '- C .~. .. ' '. ..; . ~ diseases. Antibi$t~cs'are efiemicals foreign"te'the`bodyity produce '-- : undesirable side effects, they are useless in most cases against virusesarp~ _ and certainly in most cases against cancer. But here's a great opportun- ity - we have this splendid antibody system`that Nature has evolved. The' ' human body uses antibodies mostly effectively and, unfortunately sometimes'.: too iittle,and too late, not only against bacteria that antibiotics attack,': but.aleo against viruses and perhaps, against cancer cells. But what are antibodies? They are special protein molecules specified by DNA and RNA,'"'Ftx just as we've been hearing for two days. Now one of our dreams is that sometime down the road antibodies will be a new class of therapeutic agents. A specific, different antibody would be designed for each diseaee,; If the world antibiotics market is approximately ten billion dollars, .rhat- - - - do you suppose the world market will be for antibodies if they work?,: The first class of compounds I said we could manufacture are productsn, ; whieh were previously uneconomical.The second class we can manufacture are products like human insulin end antibiotics which were previously impossible. The third class are compounds which were, up,to now, abso ~~i ; lutely inconceivable. I'll name two examples. .,; T' r 4?~j }Y - -t... .. . _... . . -." Ii'.~ ! T..r,. The firet exemple,.,- a truly thermophilic biological,~nduetrial pro a.: cess:. I think the chemical engineers in the audience would tell the bio-,' ~ logists, "We want a biological process that could operate at 50, 60, ors:.~. 70•C. We have many reasons to like such temperatures from a chemical ,. engineering point of view." But, until recently biologists would have had to reply that virtually every protein becomes denatured somewhere between 40 and 50"C, and that DNA strands "melt apart" somewhere between 60 and 70"C. How in the devil are you ever going to make a bug that can function at temperatures where thermodynamics tell you that those hydrogen bonds so crucial in biology won't hold these molecules together properly. Is it a lost cause? I think you'll agree that it is inconceivable that we can ever expect to start with an organism discovered in the soil and happily func- tioning at 25•C, and then by a series of mutations change it to function well at 70•C. Remember, each desired mutation is very rare in itself and, - the probability for the thousands of mutations which would be needed occur- ring without totally injuring the DNA is so low, that we can say it will never happen. another way to tackle the problem.`~ But don't give up:~.;Theze`Se now Why don't we mutate the'one enzyme that we're interested in, let's say it's glucose isomerase, to the point where it will indeed maintain its three dimentional structure, and function at 70•C. That's conceivable.` The " state of the art today,is that we could search and find that mutant. Now, we have glucose isomerase that will function at 70'C., existing in a cell.~~ Everything else in the cell likes 25'C. Next, we go to Yellowstone NationaL, Park and isolate some of the organisms in those bubbling mudpots. One thing is for sure.- Anything living in there has somehow, and we really' ` don't care how, solved the problem of those hydrogen bonds in the DNA and in the protein and everything else. Wouldn't it be nice if these organisms ' had glucose isomerase activity? But chances are they don't, if Murphy's Law operates.' What are we going to do next? We'd like to take that organism in which we mutated that one protein, glucose isomerase, to a will_ingness'to exist at 70"C, and mate it with Yellowstone bugs put in the "` same tube. 'As Don Glaser said, they find each other awfully quickly. The capacity to make glucose isomerase will be picked up by the Yeilows_tone bugs. Until recently, if that didn't work, the ballgame was over. And of' course, with Murphy still around, the bugs won't mate. They're in dif- ferent species. By definition, things that are in different species don't mate. >There are mules and there are ligers and there are tions, and a few`•' other irierd exceptions to this rule but, by and large, you cannot cross species boundaries with sex. And none of the other processes wprk either. The DNA can't be transferred (no traneformation).. None of the viruses that -'- infect the one infect the other (no traneduction).1 But don't give up.: We now have recombinant DNA. •You can go into your first bug, chop off that piece of DNA that codes for the 70•-tolerating glucose isomerase,,and

, stitch 1E into your YeTIowstone bug ~iand'there•you'go...We've•found a way to cross species boundaries with che_mistry. Chemistry has replaced sex And that opens up a whole series of things that were previously inconceiv- ,-. able. My second example is a'class of compounds that were equally inconceiv-" able in the old days, and I think actually something very real nowadays. That is the concept of making a synthetic gene to make a synthetic protein - to do something you want. It's an ultimate case and it may be routine a hundred years from now, but with things going at'the rate We've been seeing# maybe it'll be only ten years from now. Let's say you want to convert chemical A to chemical B. You know what kind of a reactive site you need in an enzyme or in a catalyst. You go to a computer, and find out what sequence of amino acids is going to create that three-dimensional shape: * Then you go back and specify just exactly what it is you want to have in ; the blueprint that's going to specify that specific enzyme formula. Or, in'' a very much more trivial example, you can specify the optimal combination of amino acids desired for nutritional purposes in a protein made by a microorganism. What I'm saying now is, you go to the DNA, you specify the ' code, and you know what protein, with what characteristics you're going to get. That reminds me of another little piece of elegant molecular biology.. It was mentioned yesterday that E. coli's genetic map is only 10% run into,o the ground. Six months ago it was announced that the entire map of a virus has, in fact, been run into the ground. The virus is called +X17d. It has nine genes including stop and start signals. Not only has the sequence of the amino acids been determined for the protein product for each of those genes, but the exact sequence of the nucleotides in the coded language of the DNA of that particular virus has been determined. That's an enormous string of nucleotides. I have a reprint here of that work if anybody wants to see it. It took three pages in Nature just to spell out the coded message. And that's what it is - s coded message. It's just chemistry. And the solution is not as obvious as it might first appear because there are certain redundancies in the code, so that knowing the amino acid sequence still leaves certain questions unanswered in terms of what the DNA sequence might be. But it's all been worked out. And it's just chemisty. 3f you call a virus a living thing, we can now, in a test tube, make the DNA for that living thing - and that thing will live. It will go in and infect E. coli and make more virus then before. But there's more to be excited about it is a piece of additional elegance that will appeal to those of you who are engineers. It was discovered to everybody's aston- e'4 .*`" Sshment that the information ie multiplexed. Recall that when considering a certain sequence of DNA and assuming that the code is read out three letters at a time, there's a question of reading frame. Where do you , s` start? It turns out that there are two different proteins specified by one ~' lth of DNA and this is accomplished simpiy by shifting the readinq enq, frame one nucleotide over for starting to read three at a time. This givee f r.' you an idea of the level of sophistication and understanding that is presently available to us. t~~ ~z^ I say "us." I'll return to my opening thaae.,_In the past it's been i ~~,~~that we're the biologists and you're the chemiste.„ I don't really think ;' ~ i`that we should think of things that way. . Let's simply say that chemistry #~4 its challengeal but we can accomplish more if we think ' and biology each has ~~ of this as a paitnership. . < ~.. ~i There have to be changes in reactor design.,;:Where ie it written that b ` the•way to make biological systems work industria3iy is to take a bathtu ='+and stick a propellor in it? Yet, that's the state of the art.- Now, again iI'm being very unfair because some elegant work has been done with airlift fermenters and with other configurations of the bathtub. But, clearly, nobody has gone back to ground zero and said, "Let's forget about enclosed ;:'tS vessels altogether. Let's just totally rethink this thing." And, with the "'r•new biology, and with new organisms being developed qpecifically with some 4induslrial process,in idnd;rlha.dasiqn o£;the..final-plantishoulE.=be,:I"V think, in our mindi.from the very outeet, t.-.lt;- We've heard arlot of talk about continuo4s proceasea:..Certainly„ as. ~we direct more and more of our attention to fundamental chemicals rather than to very high priced antibiotics, it makes more sense to think in terms ! of,cont_inuous process design. with its continuous fermentation process ICI ;i,is one of the world leaders in that particular respect. I don't know of '-`any antibiotic which is currently made that way. In fact, it has been . . stated by one of the world's authorities that one of the reasons that °"fermentation technology has not moved.forward more quickly has been that the markups in the antibiotie world are so large that incremental costs are simply not important enough to demand the attention that they would command were manufacturing costs thirty, forty, or fifty percent of the final cost, }`of the product. Now, we also Aeard'a`bit-about immobilized enzymes and immobilized rs- ~ ~ calls. Quite clearly in terms of economy, and optimum utilization of the itill ih fcrca eementsn te'system, there's great advantage to be had here. A- '` '.recent review article mentioned that in one process for making glutamic acid there was a 50% increase in return on investment - due in that case to lower capital costs - by immobilizing the cells and, in that case, treating ' them as a bag of enzymee* cofacto;s and so forth. (In most other senses, `•that old generalization of a living cell being a bag of enzymes is a ter- rible oversimplification' , will just list a feN othei.things that might be_eddresaedby' i ' .usn Ypartnerahip You've heard of most of them before ' Why not use zecombinant.` .. DNA technology to stitch the DNA forllltiit itof ceuase acvyn one o our best ethanol producers? "Cattle dung, as you know, was examined by General ` Electric to be recycled for use as cattle food. Maybe that's not such a good idea, but reexamination is the name of the game. Maybe dung is a good substrate for ethanol production - the volume of wastea from chickens, pigs _ and cattle is enormous. ..; . •. , . • I think you all know about the situation_in Brazil where, by edict, the government has targeted by.1980 that 25% of all automotive fuel shall be ethanol. From sugar, from cassava, whatever. But it's often been calculated that 2% of the land mass of Brazil could, if cassava were grown on it, wipe out the need for oil imports for the whole country. These „ kinds_ of considerations are now being examined very seriously. ,' Nobody here has talked about metal concentration and recovery from metal ores or slag heaps. Bacteria do this. Nobody has talked too much about cancer. It's the thing that's put into every grant application, and it gets a lot of newspaper attention. But these techniques, after all, ';were developed although the public doesn't to ralize it to fid , seeme,n , safer and more precise ways to study the causes of cancer. The public, however, is becoming aware of our new biological capabilities. I intend to P come back to the irony of.the present public controversy about recombinant f!• DNA i ft. ~ n aew momens. .,. we` There are still sqre.tarqeti for'our mutual at[ention.'';see excit- ing headiines almost weekly about breakthroughe In recombinant DNA accomr. plishments._ Almost none of them, however, secm to be focused on the need for a workable industrial process'down the road if the benefits are ever rgoing to be translated into benefits to the public rather than to a scien- tist seeking the emulation and prizes from his peers. Specifically,' at Cetus, we feel that insufficient attention is being paid in the early ..: - - - stages of the work to designing the actual organisms which will`be used in "' '`production tanks to manufacture insulin, antibodies,'alcohol, and so forth. Suffice it'to,say here that it's not enough that these microorc~anisms demonetsata genetic dexterity and the willingness to do a job ~hich is-';" <`conspicuously different than what Nature designed •them to do in the first place. - It is equally important from a commercial point of view, perticu- larly for large volume products, that they do so safely and economically, using food sources whose supply can be sufficiently assured to justify the design and eonstruction of dedicated manufacturing facilities.

- _ , . ~;~., Sometimes the same genetic engineeringapproach may'have two'differont"- . -yccs. ror exampie, an organism wnose energy consumption is tightly I coupled to.its growth rate would be an excellent candidate for use as a . _ single cell protein source The sam r i i ~ . e o gan sm n which these two functi ons ~ are uncoupled, such that it produces a lot of carbon dioxide rather than starch, fat and protein, might be very well suited for waste treatment and pollution control. or t,~iincieasi " c the , timebeinganywayseemstobea zedherring,"notwithstanding its frequent mention in the public press. The dream that a nitrogen fixation {~ capability can be transferred from bacteria into the permanent genetic rs'message of a commercial crop plant, which now requires fertilizer, is at present an objective beyond our reasonable grasp. Here, there are some technical hurdles which, though pedestrian, seem difficult to aurmount. More fundamental, perhaps, thermodynamics may be against it, and even recombinant DNA can't violate those sacred laws. But, perhaps, even this: example makes the point that each of these opportunities will be examined - on its own merits and in rational terms with sophistication which was virtually non-existent ten years ago. I'd like to close with some philosophic observations regarding the public policy issues - the politics of regulation of work with recombinant DNA. It is my personal observation that the scientists, the academic in- stitutions, and the companies presently involved in this work are behaving far more responsibly and prudently than is generally perceived by the public. what has happened is that an emotional issue involving concepts R2wnrcn cne puoiic unaersLanas oniy aimiy, but responds to strongly, has been ;~- identified by political activists as providing them with an opportunity to - express their views before a far more interested audience than they have ever had before. They charge that industry intends to "exploit" recombin- __~ .~_ - ....- - •. ••....-.... - .. .. . awyivi..caa.p aucyacc using recombinant DNA like a drunk uses a lamp post -- more,for support than for illumination. Even some of the strongest activists concede now that there is little hazard in this work. Indeed, no harm has ever been demon- strated to result from it. However, they, quite rightly I think, do iden- ' tify as a major issue which society should discuss, the question of how imporkant policies are decided, particularly when they have highly techni-. ,- cal overtones which are difficult for the public to understand. Our diffi- culty has been that their attempts to involve the public have politicized the dialogue that scientists hpve a great deal of difficulty in dealing , x. rationally with the polemics. It looks now, however, as if the voices of 1 1 reason will prevail and recombinant DNA will be regulated in a sensible ~ manner. And the activists will go on to use some other issue to exemplify;.. - !~i;~ their cause. Indeed, that is fortunate, because it would be a terrible shame if a c- ,~'c ;t " t they can ~.acientific breakthrough whose ramifications are so immense tha ~4 be described only feebly except by comparing them to those of harnessing nuclear energy - it would be a terrible shame if this technology, dis- covered in the United States, were to be senselessly crippled from pro- - ceeding ceeding here while the rest of the world hurtles forward with it, both scientifically and commercially. That is what this meeting has been about.. Let us hurtle forward together. Let the new biology be developed by those people with the most' natural interest of all in that development. We feel that many of the people in this room have that legitimate interest. We came here hoping to i.get your attention, striving to infect you with our enthusiasm, and confi- dent that we can move forward in a partnership to do wonderful and exciting things. ~~ooo"

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