 I'm going to tell you a little bit about David Green performing this lectureship, this name. I'm familiar with his work, or I was familiar with his work on beta oxidation of fatty acids, but I didn't know that much about him, so I did a little bit of research, and I just liked it, you can know for sure, the remarkable biochemist, he was an American biochemist, he was born in New York City, trained in Cambridge, and then brought kind of state-of-the-art entomology back to the U.S. in the early 40s, and was first a research fellow at Harvard, and then was an instructor and later an associate professor at the medical school, the Columbia Medical School. And he was invited to Madison to really start the first, or to start the first research group at the Enzyme Institute. And I'd like to read this quote to you, this is from his obituary, which was coded by Helmut Weiner. He said, Green therefore was a logical choice to be invited to head the first research group at the Institute for Enzyme Research at the University of Wisconsin in Madison. An institution conceived by a far-sighted biochemist in Madison, who felt that the tradition of the great biochemical research laboratories of the war-torn Europe, such as those at Cambridge, Berlin, or Heidelberg, now had to be continued in the New World. This is the great part. So Green accepted this post at what, at that time, must have appeared to have born New Yorker, educated in Cambridge, as a far outpost of civilization. At Madison, or initially at Columbia, then at Madison, he did really pioneering work on fatty acid-grade oxidation, which I mentioned, but also the design of ways to methods to dissociate, purify and reassemble enzyme systems. He worked on oxidoid reductases. He worked on the role of non-heamed iron and copper in the respiratory chain. And he discovered the role of ubiquinone and mitochondrial respiration. But he was quite the pioneer. And I'd like to introduce another pioneer today to you, which is today's speaker, who is using COSLA, who is the Wells H. Rausser and Harold M. Anakrim Professor in the School of Engineering at Stanford University. Professor COSLA is the Chair of Chemical Engineering and has appointments as well in chemistry and biochemistry. He received his PhD with John Bailey at Caltech and then was a postdoc at the John Innes Center in UK. Over the course of his not very long career, he's received many awards. Those included from the American Chemical Society, the Pure Chemistry Award, and he called it the Scholar Award. He's a member of the American Academy of Arts and Sciences. And in 2009, he was elected a member of the National Academy of Engineering. I know Shayton's work best. His research on the enzymology and the re-engineering of polyethylene synthesis. These are modular enzymes, modular enzyme complexes that synthesize polyethylene and if you're not familiar with polyethylene, this is a class of natural products that have a wide variety of biological uses and pharmacological properties. So with that, I'd like to welcome Shayton to Wisconsin-Madison to give a seminar today discussing the modularity of polyethylene synthesis 20 years old. Shayton. Thank you, Jeff. Thank you for your... Can you hear me, Patrick? No. No. Is that better? No. Now, thank you for the very kind invitation to give the David Green lecture and to your colleagues in the department for giving me the opportunity to honor somebody who clearly was a great biochemist. My connection to David Green is basically through the very close similarity between fatty acid metabolism and polyethylene metabolism, but other than that, obviously, I do not overlap with him at all. I did, however, at the time when I accepted that time invitation to give this lecture, I think it was early summer, one of the first things I did was contact another colleague who was Dick Hutchinson, who has been a long-standing source of inspiration, advice, friendship, and a lot of really outstanding science over the years. And I suggested to him that we use this opportunity. Hutch was a die-hard Wisconsin sea, and he would never cease to use the opportunity to brag about Wisconsin winter. And since I knew I was going to be here in the heart of winter, I suggested to Hutch that I would come out the day before so he and I could spend the day by your beautiful lake and he could introduce me to some of the pleasures of two foot thick layer of ice on a lake. Unfortunately, as you also know, that did not come to pass because earlier this year, Dick Hutchinson passed away of metastatic cancer. And so with the permission of your faculty, I have chosen to dedicate this lecture to Dick Hutchinson. It is not the typical lecture I did, and for those of you who came here wanting to see the latest data on the enzymology of other types of cases, I apologize. It may not be here. But I thought nonetheless this could provide a useful teaching moment for those of you who may be interested in the recent history of a field that obviously has been very close to my heart over the past 20 years but was certainly defined by this one giant in the past 20 years. As I was trying to think of how I could prepare a lecture that would be both informative to a sophisticated biochemical audience like this but also allow me to do what I felt was appropriate on a day like this, I thought back to another similar occasion recently a couple of years ago when my colleague Arthur Cornberg passed away. And the faculty in the biochemistry department decided to do a very unusual celebration of Arthur's science which in my experience of having attended some of these post-fitness celebrations was unquestionably the most enjoyable of those celebrations. What we basically did was we took an entire day out of our calendars and spent the entire day back with the students huddled in a small lecture hall and did a journal club on 20 of Arthur's papers over the past six decades of his science. And that was perhaps one of the most enjoyable experiences and so I decided I would use that theme to honor Hutch today. What I have done in my lecture for my lecture today is chosen four or five papers from Hutch's past 25 years of science and I would use them to motivate things that we did in our labs and how what Hutch did in his science influenced the direction of our own research. These are the four natural products that Hutch worked on over the years that I will be drawing from his literature on. And so for those of you who've never seen an aliquitide before you're looking at four very beautiful examples of aliquitide in a biotics right here. So the first paper I picked was a paper that Hutch published in 1989. It was one of two back-to-back papers that came out where one of them was from Hutch's lab and it was actually quite informative of Hutch's style of doing science. He really did believe that even when one competed in science science won when people collaborated even as competitors. And this notion of putting a back-to-back paper with his arch-competitor here but also a friend David Hockwood who was Michael's doctoral mentor was very typical of Hutch's character. This paper basically broke. It would be an understatement to say that this paper broke and broke. Because it did a lot more than break no well. It brought at least 50, maybe 100 years of natural product chemistry around polyethanobiotics. In one paper it brought it into the world of biochemistry. And more specifically what Hutch did was he had cloned, he went five years before this. He went to Norwich, England to learn genetics. Long before most chemists could even spell genetics. Hutch made a leap into the world of genetics and did so with quite a bit of flair. He got back to medicine and launched and realized that the only way he would be able to make further advances into biosynthesis of the tachycinomyces which were antibiotics he was interested in for a while was to look at the genetics of these biosynthetic pathways. And this paper basically described the cloning and sequencing of the core polypetyte pathway within the tachycinomycin biosynthetic pathway. And specifically what Hutch observed in this study was that the enzymes that made this polyfunctional aromatic antibiotic called tachycinomycin were basically very close relatives of the enzymes involved in fatty acid biosynthesis. Specifically he observed a small protein called an acyl carrier protein and he observed a condensing enzyme that forms a carbon-carbon bond between an electrophilic iris and the eucrophilic malonate extender shown over here that would be a condensing enzyme or a chelosynthase. And he proposed using the well-known chemistry of fatty acid biosynthesis an analogous pathway that would build up the carbon skeleton and subsequently cyclize it into the first isolable precursor of the tachycinomycin epilates. And I've highlighted this sentence over here because I think if there was one sentence in this paper where that demonstrated the man's vision about where this field was headed it would be this sentence where he was persuaded that this would be the beginning of what today we know as multi-design, assembly line antibiotic biosynthesis. And I think this was really far reaching back then. It would not be an understatement to say that this paper together with his companion people were the two papers that inspired me and I decided to take my own career in the direction of antibiotic biosynthesis coming out of graduate school. Hutch was persuaded very early on before there was any direct doubt that these enzymes would be working like a complex. In fact, this is a paper with your now colleague Ben Shin who was a postdoc with Hutch that illustrates that about as vividly as vivid gets. He basically characterized this system, this polyketide synthase as a heterodynamic ketosynthase onto which this little acyl carrier protein would come and set to be able to go through a series of reactions. So you've got a thiolz on a banditine arm over here. You've got an active type cysteine over here. At that time he had drawn about where this nucleophilic transferase was but that's a relatively minor detail in this overall pathway. But basically he had gotten these two active sites right on the money and he had predicted that these things would come together to form this complex. You would have your malonate attached to this carrier protein dial. You would have your first acetyl unit anchored on the active site over here. Carbon-carbon bond formation would happen and now you would be set up to go through this cycle again as many times as it took to build this depaketide chain which would then be cyclized, aromatized, and eliminated from the system into this compact. So this thing that these systems would set together and assemble into complexes, into assemblies and do this assembly line biosynthesis was very close. He was absolutely convinced by the early 1990s that that's how these enzymes work. To my knowledge this represented the first feeble direct evidence in the literature for the existence of these assemblies, these covalent complexes. This was an experiment that your dryer did in my laboratory in the late 1990s where essentially what he did was he titrated in an Apo acyl carrier protein a carrier protein without a banditine part and showed that it was a competitive inhibitor of the polyketide synthase with a KI on the order of the KM, the apparent KM of the holo protein. So when York sent this paper in to JBC there was other data in this paper too but one of the reviewers just did not like this paper and the fact that there were only three points on this graph that we used to calculate the KI and there was no 4x4 plot and so on and so forth and there was a very good chance that that paper would not have been accepted in the literature but in his characteristic style Hutch wrote the second review of the paper and he anticipated what the first reviewer would say and he preempted a lot of what the first reviewer's concerns were and then he went on to explain why it is so hard to do these experiments because you get such little quantities of this polyketide synthase out of these enzyme systems and he signed his review, Hutch, as he often did and I think it's fair to say that that paper was published because of Hutch's generosity of spirit at that point in time of course several years later, Yi-Tang came to my lab and did the experiments the way cell-perspecting biochemists would want them done he took the actin-arodin-optic-ketide polyketide synthase the tetra-synamycin synthase titrated in a whole bunch of different carrier proteins and showed that there was specificity in this chemistry in this biochemistry for the actual protein part you can just draw your attention for example to these catalytic and parameters and you can see that for a given chino-synthase as you change the identity of the carrier protein even though the rest of the chemistry that it's doing is identical there are at least two logs worth of differences in the rates of the reaction simply as a function of the identity of the protein so ten years after Hutch's insight that these things were assembled the case has been quite well there are many other experiments in the literature by now that made the case so fast forward today this is how we know the pathways like the here I have shown the catalytic cycle for the pathway of the actin-arodin-symphase that was the companion paper to the tetra-synamycin synthase paper that I initially alluded you to and we understand this pathway we have crystal structures, high-resolution crystal structures are pretty much or NMR structures are pretty much all the proteins in this pathway for those of you who may not be familiar now I can walk you through perhaps the pathway a little bit better you start with this carrier protein over here with its pentatine-armed thiol there is an enzyme that is literally borrowed from the fatty acid synthase a malonyl-coeacin-transferase that takes the malonyl group from coenzyme A attaches it on to this carrier protein associated in a dedicated manner with this polyethycythesynthase and now you are set up for priming the system this is the one thing that Hutch didn't quite pick up from his earlier analysis but soon recognized later on that the priming of the system essentially happens by g-carboxylation of a malonyl extender in the active site of the thiol-symphase so essentially the initiation reaction is involves the entry of this nucleophile into this active site since there's nothing for a carbon-carbon bond formation there's no thioester attached in this active site at this thiol you end up g-carboxylating this it protonates, it quenches to an acetyl group the acetyl group now transfers onto the thiol and now you have a crime keto-symphase you have another copy of this extender unit that comes in that's generated by the same fast pathway over here you now have a nucleophile and an electrophile sitting next to each other in the active site the carbon-carbon, this carbon-carbon bond forms you have a data key type a beta-ketoester that forms and importantly, and this is the key observation that allowed Hutch and us to be able to mechanistically differentiate the assembly line synthesis that I'll be talking about in a little bit where this transfer of energy gets to this recycling that this carrier protein leaves after it returns the ketide unit back to this active site a new carrier protein comes in with one more, you have elongation and finally you have the chain that forms and that's what leaves it so this pathway by now is very well established notwithstanding the highly reactive nature of these intermediates as these enzymological investigations were going on the pioneering observations that Hutch made about the chetrocynomycin synthase the associated enzymes as well as the companion observations that were made by looking at the generics of the active eroded polyketides synthase allowed Bob McDaniel in my lab my first graduate student to start doing comparative studies on the molecular recognition features of these different enzymes and over a relatively short period again thanks to the generosity of Hutch's lab for having given us access to a grant and this day and age you never think about these things because when you need a gene you just call out some DNA synthesizing facility and next day your gene shows up that then the only way you can do these scientific experiments is if you add good enough relationships with the people who originally characterized them and in that context I'm extremely grateful that Hutch was able to give us access to these genes that he had maintained in the clone and they could do a comparative molecular recognition analysis of the active eroded enzymes the texasenomycin enzymes characterized their chain length specificity, the keto reduction specificity their cyclization specificity for these reactive polyvated keto intermediates how they aromatize and so on and in the course of these studies Bob McDaniel was able to develop some kind of chelicin design rules for these kinds of molecules these design rules had obvious engineering implications because by then the third and the fourth gene clusters of this family had started to be cloned in sequence and one could actually visualize how one might be able to use those design rules to do rational engineering of polyfunctional aromatic compounds and so here is an example of a small library of compounds that Bob McDaniel had come to made in my lab starting with Hutch's active eroded polyketide synthase genes Hutch's texasenomycin polyketide synthase genes and another class of a related polyketide synthase involved in phenylison biosynthesis where systematically mixing and managing these genes one was able to access a variety of compounds that including something that really didn't have equivalence before in the natural product literature so I think that's one example of how the early insights from Hutch's lab were able to influence not just mechanism but also engineering of these kinds of anabolic pathways what also became quite clear from these kinds of experiments was that the critical parameter that nature was setting in these kinds of polyketide synthases was the number of cycles a given polyketide synthase would iterate in order to make a highly reactive polyvated ideal chain so Hutch's polyketide synthase iterated nine times with 10 malonal acyl carrier proteins to make these kinds of compounds see 20, 20 carbon compounds Hutch's polyketide synthase iterated eight times with eight malonal coenzyme A equivalence to make these two 16 carbon chains cyclized differently but nonetheless they had a very strict so straight specificity and there was an important question that the field was struggling with what was controlling this chain length specificity at that point in time so very early on in these experiments again Bob McDaniel did an experiment in my lab when he took Hutch's Keto-Polyketide synthase that made technosinomycin Hutch's polyketide synthase that made the actin rodent skeleton and basically scrambled the three proteins the Keto-Synthase, the carrier protein and there was a third protein in there in this minimal polyketide synthase that nobody really knew the function of back then but that he basically made all possible combinations of these hybrid polyketide synthases and characterized the chain lengths of their corresponding products and what he observed through this experiment was that for all the products that he was detecting the chain length specificity basically trapped with the origin of this subunit so for example if you take, let's see this one over here this particular one this is basically the technosinomycin polyketide synthase but this particular subunit or protein comes from the actin rodent synthase and that makes a 16 carbon chain and so Bob basically called this protein with David Appel's blessing the chain length factor and so when the next time I had a chance to meet with Hutch I introduced to Hutch the concept of a chain length factor of course by then Hutch had read this paper and with his characteristic big smile he looked at me and said I don't believe it and I said why? he said see this column over here you didn't get a product in here when you combined the technosinomycin chain length factor with the actin rodent minimum polyketide synthase when you were able to make a 20 carbon polyketide with the technosinomycin enzyme over here come and talk to me and he was right in fairness in order to be able to definitively call this subunit the chain length determining factor we should have been able to do both these experiments and Hutch but this is a characteristic trait of Hutch he would never settle for anything short of perfect well that was a sufficient challenge to a young upstart assistant professor like me that several rounds of students and postdocs spent considerable portions of their PCs and postdoctoral studies trying to address this specific problem and it was only about 10 years after those experiments were done when Cheryl Tsai and Yitang were both in my lab they were able to develop an accurate enough molecular model of the active site of that herodinary ketosynthase to be able to predict what were the precise residues that were lining this pocket that were dictating the chain length specificity of this enzyme they made the appropriate use and indeed this was it turned out exactly as they had predicted so here is the wild type actin-erodin ketosynthase that makes a 16 carbon polyethype chain here is the trace of the wild type tetrasynamycin synthase that makes a 20 carbon polyethype chain and here is the trace of a mutant where only two residues as predicted by Yih and Cheryl's model in exclusively residing in this chain length factor subunit so both these residues are part of that chain length factor were changed to their counterparts in the ketosynamycin synthase and now you can see the major product over here it was a 20 carbon polyethype synthase so basically this is the actin-erodin synthase with just two residues borrowed from the tetrasynamycin synthase both in that chain length factor subunit and it predominantly makes this C20 product and importantly this time they were able to do the reverse experiment they were able to introduce just one residue from the tetrasynamycin synthase into the actin-erodin chain length factor and have it predominantly make the correct chain length so this was, we were really excited about this by then Hutch was no longer an academic Hutch was the vice president of research of a biotechnology company in the Bay Area that was developing polyketide drugs called cosane biosciences Hutch had better things to do than read Jack's ASAP by then but nonetheless Hutch did break up every morning and look at Jack's ASAP when this accelerated publication came out when this communication came out in Jack's Hutch was the first person to send me an email saying I now believe it and that to me meant a lot that was a very important acknowledgement from Hutch and it did mean a lot especially given that he had very little at that point to do with enzomology here's another paper of Hutch's that goes way before the field by the early 1990s it could have been even earlier but certainly by 1919-1991 Hutch had gotten very excited about the possibility of studying doxodemus and the biosynthesis of doxorubicin and he was motivated by that ironically given how his life came to an end by the fact that doxorubicin even though it was a frontline he was therapeutic for a variety of cancers was an extremely expensive agent and he deeply believed that studying the biosynthesis of doxorubicin could lead to the emergence of better technologies to make doxorubicin and indeed his life did develop some very useful technology to be able to enhance the productivity of doxorubicin that was subsequently licensed to Pharmacia which was the primary producer of doxorubicin for improving the yields and productivity of doxorubicin but in the course of his studies in doxorubicin biosynthesis Hutch made another important discovery with regard to polyethype synthesis he discovered that and this was I should say not a serendipitous discovery this was anticipated he had anticipated so this is the structure of the real doxorubicin he had anticipated that this hydroxy ketone moiety was the result of an unusual variation in the catalytic cycle I showed you earlier on so this is also a depaketide backbone but it nonetheless comes from one so that mechanism of decarboxylative timing that I explained to you earlier on in the context of tetracytomycin could not apply in this system they had to be something to actively suppress that decarboxylative primary mechanism he didn't know what it would be but nonetheless when he did the genetics on this pathway he discovered two critical enzymes and basically they turned out to be a dedicated initiating module for polyethype biosynthesis and this was the first of several examples subsequently a number of polyethype biosynthetic pathways were looked at including in my lab the biosynthesis of this family of estrogen receptor antagonists called R11-28 antibiotics and that also had these unusual appendages and indeed it became clear by looking at these and a variety of other ones that nature had developed this was sort of the first step in nature it was armamentary to start evolving to more and more complex polyethycythesis in the sense that if you saw the tetracytomycin synthase as basically this pathway over here that just goes round and round several times to make a backbone what nature had done in the context of Dr. Rubison or R11-28 in our case had tacked on to that an additional module of polyethycythesis enzymes where now the catalytic cycle starts with the priming of this dedicated ketosynthase with some random coenzyme a precursor that exists in the system not randomly as dictated by the molecular recognition features of this enzyme you now have, in Hutch's case a procurial enzyme adduct or in our case a variety of other depending upon what the identity of these precursors was this goes to one round of elongation followed by a full set of reductive enzymes that reduce this into an alkyl chain and then this, the kerala protein that's part of this module passes this alkyl precursor on to that aerodynamic ketosynthase that I was talking to you about earlier on and now this guy takes on those two as many rounds as its chain length, specificity factor dictates and out comes the desired problem so now you have the beginnings of a mechanism from nature that allows you to do something that would be chemically, virtually impossible to take a polyketone and a biodexane like this and be able to functionalize an inert methyl group in an otherwise functionalized polyketone chain to make some kind of other modification in this position this would be a chemically, virtually impossible thing to do in a molecule of this sort but nature had figured out how to do it using the polyketone principles and so again the question came up how could you use the insights of this kind of a bimodular synthase to be able to develop rational design rules for being able to take an antibiotic of this sort or a natural product of this sort and re-doselectively functionalize an inert methyl group over here so the answer to that came in two steps the first step was to be able to get the recognition features of the carrier proteins and the condensing enzymes set up in such a way so that you could have an initiation module that would talk to the elongation module that makes this molecule so you've got an elongation module over here with a helo reductase that makes this molecule you want to append on to its front end an initiation module of polyketone synthase subunits so that that would feed in the desired functional group the alkyl functional group that you want to put in instead of the methyl chain and that would be taken up over here but that's the only thing you do you recombine two modules where nature had only one you're going to hang that with a molecule that does have an alkyl appendage but it's not the analog you were looking to rationally produce which would be functionalize over here and the reason for that is because the chain length factor of this module is reading carbon atoms not the number of condensation cycles that are going through by adding this extra fluff over here you have gotten this polyketone synthase to truncate the number of condensation cycles it is going to do so if you do want to design a molecule which is functionalized over here you want to make two changes not just figure out how to add boltons of front end this initiation module to here you should be able to replace the chain length function over here so that now you have let's say you're going to introduce a butyral unit or a pentanoil unit over here in place of an acetyl unit you now need to have a chain length function over here that would be a deca ketide equivalent instead of an octa ketide equivalent so you need to get two extra ketides in that chain length rocket and you can essentially have this molecule this alkyl group incorporated the bulky group and at the same time go through seven rounds of condensation so that the reactive portion of the molecule is set up for cyclization and modification in this way so basically the principles that came out of Hutchinson's studies on the doxorubicin synthase had direct engineering implications if you will rational molecular engineering and since then a variety of modified compounds have been made some of which are shown over here where you can systematically using this principle modify with your favorite alkyl groups what would otherwise be an invariant method in these molecules I've been for time do I have about 10 minutes more? okay, let me tell you one more short story that I'm going to tell you about the experiment that remember Hutchinson was born so by, I don't know Ben, you're going to have to correct me if I'm wrong but by about 1997 Hutchinson was excited about a new antibiotic that he was interested in what, an old antibiotic but he was excited about studying the biocin which is called Frederickomycin I still remember the email he sent me and when he decided he was going to launch into this he called that email he had this characteristic tendency to use very graphic one word or two word titles to his emails most of us when we're writing an email to somebody, what do we do? we struggle to find the right title for an email and just put any garbage that we can find in that email header knowing that nobody reads those anyway but Hutch is to find ways to annotate his emails with very interesting titles this title came to me as big ketide it was a word that told me exactly what Hutch was going to do he was going to clone and characterize the polyketide synthase that made the longest damn chain that nature could ever make so he had looked at done the retro biosynthetic analysis on this polyketide pathway and he was persuaded that this could derive from a 15 acetyl equivalent so at C30 polyketide chain and 15 malonal equivalents and therefore he was going to call it the big ketide of course shortly after he started studying this pathway he moved on to greener pastures California or not really green anymore but she moved on and this project they follow until your colleague Ben Shen came here and had the courage to pick it up and study this pathway in all its glory and so about probably those 10 years after he started studies on this pathway then together with Hutch about the seminal paper that characterizes the Frederica Mycin biosynthetic pathway I have nothing to say about the Frederica Mycin biosynthetic pathway but it did influence something that's a very thought provoking experiment that's been going on in my lab if I'm a completely different direction about three years ago we got interested in this polyketide that was reported by a group of researchers at SanctoLabs primarily because of its interesting pharmacological properties it is a specific inhibitor of an enzyme that plays a critical role in the potentiation of the gamma interferon pathway and as such I'm sorry the alpha interferon pathway not gamma interferon pathway and as such presents a fundamentally new mechanism for antiviral chemotherapy a kind of mechanism that is devoid of many of the toxicological relations associated with nucleoside and non-nucleoside antiviral therapeutics and so we got interested in this polyketide it also represents a fundamentally unprecedented chemotype and that was also motivating to us and so God gives a letter and Lucia, who lived in my lab over the past couple of years young and sequenced and characterizing and rather than to express this pathway I've done all the things that nowadays we all take for granted and they have a pathway to propose for their their natural product and that starts basically with the same C-30 big ketide that Hutch's Thraderycomycin pathway started with but what's really surprising about this pathway is when you look at the gene cluster that makes a 745 to 8 up here and you look at Ben and Hutch's Thraderycomycin pathway they're basically important and identical they have greater than 75% identity in every gene down the rank except for this one gene out here which is absent in the Thraderycomycin gene cluster so in the past 20 years I can't tell you how many times I have been asked the question what good are all of these engineered oligetides that you make for what use do they they probably all represent waste products things that nature experimented with a long time ago and disposed of because they had no real utility to biology I'm not sure I believe that or I didn't believe that but it I think when you have to accept that when you don't have the data to counter somebody you've got to accept that they may have a point I think this clearly puts that argument to rest you've got one enzyme pathway one gene cluster one enzyme two totally different antibiotics both of which have two totally different pharmacological behavior basically out of a kind of gene this isn't repurposing antibiotic biosynthesis in a modular fashion I don't know what it is I think it's fair to say we would not have been here in such a short period of time were it not for Hutch's the ketide discovery let me conclude with this last paper of Hutch's because if you pushed me against the wall and said pick one paper that Hutch wrote that changed people's thinking more than any other I would probably unless I was very drunk in which case I would pick the first one that I showed you but I would probably pick this because this was a very important paper it was published in 1987 and again in Hutch's characteristic style this paper was published back to back with a similar paper Hutch was studying tylactone which is the antibiotic precursor of the antibiotic which is the macrolite precursor of the antibiotic tylacin and David Kane was studying or thronolite which is the macrolite precursor of the antibiotic retromycin and they published back to back papers that were very consonant in terms of thinking message what was the message so by then even though nobody knew what these polyketides would look like people had a pretty good idea that these polyketides were derived from fatty acid like macadazins but there were two questions when you moved from something as simple and functionally boring as a fatty acid to something as complex as tylactone this is a crummy slide in terms of resolution no offense to hyperfoils but there's only so much you can do with these horrible ACS scans from past old papers there were so when you look at a molecule like tylactone which is what Hutch worked on in this study it is not at all obvious how such exquisite and rich functionality in stereochemistry could have arisen from something as boring as a fatty acid synthase going alright and so there were two models back then that people struggled with about how this functionality in stereochemistry would be introduced one model was that you would have acetylchropionic butyronal units oligomerized in a predictable fashion by say the tylactone synthase whatever that was going to look like into this poly beta keto chain would be appropriate functionality methyl branches and ethyl here for good major and so on and so forth and then there would be a series of decorative enzymes that would come along modify this highly reactive poly beta keto intermediate into something that starts to look like tylacin the other hypothesis was nature would come up with a way that it would do all the chemistry that was required incrementally so you would have the formation of a dietetide but then you would have this stereocenter and this stereocenter as well as this alcohol functionality set at that point then you would go on to make this dietetide into a triketide again set the functionality this olefin over here and at that point then you would go on to make this tetra ketide and so on and so forth well of course sitting where you are today you know what the answer is but back then it was not at all clear what the answer would be hutch did that experiment and specifically this was the experiment he did and this was the experiment that David Cain did that was published in character basically what hutch did was he made this triketide and this dietetide this punitive precursor he basically said if the pathway looks like this then this would be the triketide intermediate and this would be the diketide intermediate of course it would be enzyme bound but guessing that it would be a thioester intermediate he was going to make a synthetic mimic of the diketide and the triketide and the question came how would he activate it so that it would be both cell permeable as well as incorporated, trans-isolated onto whatever the enzyme looked like and after that several fold starts both he and David Cain came up with using an acetyl-sustaining thioesters for this both of them got incredibly low incorporation of these synthetic precursors into their corresponding macrolide backbones but they nonetheless were able to show using isotopically labeled the intermediate so how to label this methyl group because you get a very strong NMR signal for this methyl group for this methyl group in the diketide to pick up this signal in trace quantities even though he was getting only a couple of percent incorporation of these intermediate but nonetheless he was able to show that these were intermediate David made a doubly labeled intermediate so he could look at Big's clinic in the C13 NMR and that gave him an additional 5-old or 6-old improvement in resolution and he was able to detect the intact incorporation of his intermediate importantly Hutch did a negative control experiment and this is an important message for every student to play he actually fed maybe even fed this alpha-methyl beta-keto ester because this was the expected intermediate on this pathway David didn't do that experiment shame on David Hutch did that experiment and he showed that while these intermediates incorporated well the beta-keto intermediate did not incorporate and I think this was the definitive experiment in my view in terms of breaking the breaking a log jam in how to think about macrolite biosynthesis that these assembly lines are worked from this and this is the eryconolite synthase that was originally characterized by Peter Lettig and his collaborators in Cambridge and Leonard Katz and his collaborators in Abbott but back then we had no idea that this enzyme system that the enzymes that make these kinds of molecules would look at this and there are some very telling lines that I initially grew up to show you in Hutch's paper but then realized that nobody even I wouldn't be able to read them given how bad they show but I strongly recommend that you go and read the last paragraph of this paper because if there ever was somebody who could have foretold what this field was going to look like as well as some few lines in David's paper that have other things in them too that are not fit to read but Hutch's paper is really about masterpieces it's a masterpiece in experimental design why and how did that influence my own research of course in many ways but I'll just tell you about one experiment and I'll end over here so as I mentioned these kinds of biosynthetic incorporation experiments back then were great analytical tools to study polyethylene biosynthesis and mechanisms but they were hopeless in terms of preparative chemistry when John Jacobson came to my lab in the mid 1990s he basically decided inspired by Hutch and David's experiment he was going to try and turn an analytical method into a preferably useful method and the way he was going to do it was by essentially using genetic adhering to block the synthase in its first module so now he could come along and take a synthetic diketime analog drop it into the fermentation broth but in contrast to what Hutch and David came experienced where they were competing with the natural pathway now there was no competition for the synthetic intermediate so this synthetic intermediate could go to the second module of the Erythronalide synthase and be modified and if you could do that with the natural pathway R1 is normally a natural group and R2 is normally an ethical group to make natural Erythronalide why then you could do it with buckyballs or whatever you wanted and indeed you were able to make a variety of compounds modified at the western shore of this molecule in relatively short order in quite respectable quantities so I was really excited I was on cloud nine in 1997 when John was doing this I ran into Hutch I told them about what was going on even before actually this was way back in 1996 I ran into Hutch Hutch basically thought about that I think it was on the shores of your lake outskirts although it was in summer Hutch thought about that and said what do you think would happen if you fed this trichetyte precursor to the Erythronalides synthase Honestly I hadn't thought about that experiment until then it was not at all obvious to me even after I spent a good half hour thinking about that experiment what you would get so I went home I told Jacobson Hutch suggested this experiment why don't we feed the trichetyte from Tylosin which is a 16 carbons shall we say a re-expanded macrolide relative to Erythromycin why don't we feed the trichetyte to the Erythron to your block the Erythromycin things and see what happens John voted me like only a student would I mean with extreme this case he basically walked me through all the steps that would be required to make this precursor and then he reminded me that in order to get the quantities of the modified compounds out he would have to do at least a one liter fermentation experiment which would require in the order of a half a gram of this intermediate and was I nuts or what to ask him to do such an every experiment out of here so you know as faculty we just go back in our office when our students tell us we were full of it and we tried to put our crazy thoughts out of our mind I did that Hutch called me up the next day and said so I'm going to do the experiment of us I said I can't persuade Jacobson to make 500 milligrams of that compound Hutch hung up a week later I get this shipment in my office back then there were not nearly as many constraints on shipping stuff it basically had a ground bottom flask in it that was still glowing from dry ice from having been pulled out from dry ice it was just like one of those things that had just been pulled out after God knows how many decades from your freezer that had a Jensen strike on the order of 500 milligrams and there was just one letter one piece of paper in this I mean nothing on the flap and there was just one with gold I knew exactly what it was I walked into John's office and I said here is your 500 milligrams of the tritite he was to stand forward I reminded him that the way he was going to confirm that this was truly gold was by going and taking an NMR and confirming that this carbon atom was C13 labeled which indeed he did within 30 minutes he was back saying yes it is gold and it doesn't mean the Hutchinson tritite so he did the experiment and indeed I mean this is really the kind of experiment that persuades you that nature would amazingly modulate the machine what you've got over here is a chimeric antibiotic where the left half of it is thylacin and the right half of it is re-informalizing it basically illustrates how in nature you can think through for yourself what nature would have to do in order to have converted this polyketide synthase into a thylacin polyketide synthase so once this was done John did the reverse experiment where he flipped this one stereocenter back to the erythromycin center and this compound incorporated to make an erythromycin molecule so this set up an amazing paradox for us that took about a good eight years to figure out and we now know that what this stereocenter does this stereocenter this is the Huygensen Tritite the gold relative to this compound with this stereocenter this in the Tritite has been flipped is it's not a block so the module 2 of the erythromycin phase this module does not dislike this in terms of entry this compound can isolate this module just fine in fact it can isolate the module better than it's natural substrate however this compound cannot undergo carbon-carbon bond formation in contrast Huygensen Tritite can isolate as well as enongate just fine so somewhere in this active site which does two distinct reactions in the overall erythromycin polyketide pathway lies information that allows it to use the differential recognition of this one stereocenter to influence the second but not the first reaction in the polyketide pathway of course that is the kind of mystery the next 20 years of studies on these polyketides and bases will be focused on in an ordinary circumstance this would be my first slide of my lecture because this is where this is how we currently see the 60-oxy erythromycin phase this structure this composite this picture is based on high resolution X-ray crystal structure as well as NMR information of about 25% to 30% of the entire 2 plus million dolphin synthase but because these systems these modules are highly repetitive any given active site in here is about 50% or more identical to any other homolog in the system it becomes relatively easy to build working models of the entire so this is our best guess today of what this enzyme system looks like and how building blocks go in over here and get processed to come out as naturalites and this is the kind of model we use today to motivate the kinds of experiments that are ongoing in my lab but that's a different story again I want to thank you for inviting me out here I apologize this has gone on a bit longer I wanted to go on but it would be a remiss I would be remiss if I did not one more time acknowledge the cultures a finer natural product chemistry there really has been and also a very very enlightened person somebody who has been helpful friendly visionary to a number of people self included I think this picture is the picture that I was hoping Hutchwood helped me experience at least it had been around today it hung outside his office in California for about five years I loved it every time I look at him in the thick of California winter he would talk about what like was really like in Wisconsin and he suddenly was a brilliant son of your fine state and a brilliant citizen of your fine university so with that I salute Hutch one more time and thank you for your attention two questions before we have the show taken off to the airport do you have a normal lecture tomorrow at the university after this mission you have Hutch to thank for that question where I'm not so familiar with the area how do these actually control change like these systems control change like that of molecular volume control basically volume control you have steric functional groups that are carved and active site you have an actual pure active site and basically as we take, we take these systems we're opening up the active site which is the reason why McDaniel's original experiments that Hutch complained about didn't work in the reverse way it was possible to combine put a small functional group in in a big place but not the other way around of course McDaniel didn't know that absolutely absolutely it is exactly my advice best in hand, antibiotic activity from the second open tablets that are produced well you would be okay let me just make sure I understand your question do you want to re-engineer it by just completely scrambling everything or do you want to take specific enzymes that control specific functional groups and re-engineer them to be able to introduce alternative functional groups so do you want to do medicinal chemistry on the natural product or do you want to make garbage just make every possible okay you're, it's not it's lower than one in a thousand because by now the fields probably made over a thousand natural natural products none of which are markedly superior to the current then uiotics using that kind of approach but if you do it in a directed fashion then you got a reasonable shot at making it improved in a what's the kind of in the photos they got it depends it depends what you're trying to do so probably a good example given what I just told you is to, so the actually this is interesting this experiment was done when Hutch was the research president at Kossien Johnson and Johnson wanted an analog of Rick Thromycin that had significantly superior lung pharmacokinetics what Hutch championed was the biosynthesis of 15th floor lawyer, Rick Thromycin knowing the he scaled that compound up to one kilo to a point where he was able to show in grown dog and non-human of primate that you had significantly improved pharmacokinetics and forget what the number was but the half-life of that compound compared to a parent Rick Thromycin is known how to deal with ourselves it doesn't matter so they were doing a very predictable thing because I mean very rational thing because you know quite a bit about the role of fluorine in medicinal chemistry nature doesn't waste much effort putting fluorine in natural products and so you're basically putting fluorine into the momnet so no more questions let's thank Cheyna one more time