Session 4: An Introduction to Polyketide Assembly Lines
Transcript of Part 2: Dissecting Polyketide Assembly Lines
00:00:08.07 Greetings. 00:00:09.18 My name is Chaitan Khosla 00:00:11.09 and I'm a professor at Stanford University, 00:00:15.19 and this is part two 00:00:18.01 of the trilogy of my lectures 00:00:19.28 on assembly line polyketide biosynthesis. 00:00:24.11 In the previous lecture 00:00:25.27 I introduced you to the evolutionary biology 00:00:30.15 of these remarkable assembly lines, 00:00:33.00 the chemistry that happens 00:00:34.10 on these assembly lines, 00:00:36.07 and I gave you a general idea 00:00:38.05 of what these assembly lines look like. 00:00:42.02 So, we looked at the 00:00:44.12 6-Deoxyerythronolide B, 00:00:47.03 or DEBS, 00:00:48.22 synthase, 00:00:50.11 that is responsible for making 00:00:52.23 this precursor of erythromycin. 00:00:57.21 I ended the previous lecture 00:01:00.15 by giving you a sense of what 00:01:02.18 we think this assembly line looks like 00:01:04.22 and how that insight was derived. 00:01:08.21 What I'm gonna start this module with 00:01:12.02 is an introduction to the kinds of tools 00:01:14.24 we use to interrogate 00:01:17.01 the biochemistry 00:01:19.06 of these remarkable assembly lines. 00:01:22.12 So, 00:01:25.13 these assembly lines exist, 00:01:27.27 as we discussed in the first lecture, 00:01:30.22 in relatively esoteric sources. 00:01:33.17 They usually come from bacteria 00:01:35.22 whose names many of us have a hard time spelling, 00:01:39.11 or sometimes even worms, 00:01:41.25 or often times just sequence information 00:01:45.04 that was derived from DNA 00:01:47.04 that was collected from some place. 00:01:50.16 In order to study these systems, 00:01:53.07 one of the first sets of tools 00:01:55.14 that we developed 00:01:57.24 was to be able to take these 00:01:59.15 very complex metabolic pathways, 00:02:02.12 like the DEBS metabolic pathway, 00:02:04.26 and put them into 00:02:08.00 genetics-friendly microorganisms; 00:02:10.17 hosts like E. coli. 00:02:13.21 So, today you can make 00:02:16.20 6-Deoxyerythronolide B 00:02:19.15 in E. coli 00:02:21.07 by growing it in the presence of 00:02:24.12 glucose as a source of energy 00:02:26.11 and propionic acid 00:02:28.00 as a source of all the carbon 00:02:30.05 that's used to make the product, 00:02:32.04 so long as the recombinant E. coli 00:02:34.11 contains DNA 00:02:36.26 that instructs for the biosynthesis 00:02:39.10 of those three very large proteins 00:02:42.09 that comprise DEBS. 00:02:44.18 This is, 00:02:46.16 for obvious reasons, 00:02:48.18 a very powerful tool 00:02:50.15 to interrogate DEBS 00:02:52.06 because, now, if I have a bacterium 00:02:54.29 that makes my product for me, 00:02:57.09 I can go in there 00:02:59.15 for the price of a $200 kit, 00:03:02.12 manipulate the DNA 00:03:04.12 that encodes this assembly line 00:03:06.19 and ask, 00:03:08.06 what are the consequences of this assembly line? 00:03:11.03 And these tools have been 00:03:13.02 in our armamentarium 00:03:15.09 for the better part of the past two decades. 00:03:17.20 My previous generation of lectures 00:03:20.01 talked quite a bit about these tools, 00:03:22.11 so I won't spend a lot of time 00:03:24.01 doing so again, 00:03:26.05 but these tools 00:03:28.12 have played a critical role 00:03:30.03 in our understanding 00:03:31.21 of the biochemistry 00:03:33.01 of these assembly lines. 00:03:34.17 Now, what is more challenging, 00:03:37.12 but is perhaps arguably more important is, 00:03:41.28 if you want to study this remarkable enzymatic assembly line, 00:03:46.01 you'd like to be able to peel off the wrapper 00:03:49.28 that surrounds these remarkable proteins. 00:03:53.08 That is easier said than done, 00:03:55.20 but today, we can reconstitute 00:03:58.20 the entire 6-Deoxyerythronolide B synthase 00:04:02.15 from purified proteins. 00:04:05.06 What I show you on the lower-left corner 00:04:09.09 is a protein gel, an SDS-PAGE, 00:04:12.27 that shows five proteins. 00:04:17.20 The two proteins to the far right 00:04:21.01 are the second 00:04:23.23 and the third protein 00:04:26.15 of the erythromycin assembly line. 00:04:30.20 And each of them, as you can tell, 00:04:33.12 has a monomeric molecular mass 00:04:36.01 that exceeds 300 kilodaltons. 00:04:40.01 The third protein, 00:04:41.22 which is the first of these proteins, 00:04:45.03 could not be expressed 00:04:46.23 for love or money 00:04:48.14 in E. coli 00:04:50.13 in a form that gave adequate yields 00:04:53.07 of pure protein 00:04:54.28 to study biochemically, 00:04:56.28 and so we had to break it up 00:04:58.26 into three pieces, 00:05:01.04 which are shown in the 00:05:02.24 first three [lanes] of this gel, 00:05:05.14 and purify those pieces independently. 00:05:10.08 And now you can put those three pieces 00:05:13.14 together with the other two proteins 00:05:16.03 to make a cocktail of proteins 00:05:19.25 that, in the presence of appropriate substrates 00:05:23.06 -- propionyl coenzyme A, 00:05:25.11 NADPH, 00:05:27.04 and we don't use methylmalonyl coenzyme A itself, 00:05:31.01 instead we use an in situ enzymatic generation method 00:05:35.22 for methylmalonyl coenzyme A 00:05:38.00 where we use free methylmalonic acid, 00:05:41.01 coenzyme A, 00:05:42.24 and an enzyme called malonyl-CoA synthetase -- 00:05:46.07 and so when you put these five proteins, 00:05:49.14 which have been purified, 00:05:51.13 together with all these precursors 00:05:53.26 in a test tube, 00:05:55.21 you see 6-Deoxyerythronolide B. 00:05:58.24 And what I show you on the lower right 00:06:01.07 is a mass spectrum of the product 00:06:04.00 that has been synthesized 00:06:05.29 in a biochemical equivalent 00:06:08.02 of an earth/air/water/fire type 00:06:10.19 of an experiment. 00:06:12.16 What this allows us now to do 00:06:14.24 is to probe this machine 00:06:17.05 with all the power 00:06:19.19 that you're used to using 00:06:21.24 to study your favorite enzyme 00:06:24.27 once you've purified it to homogeneity. 00:06:28.06 So what I show you in this 00:06:30.13 is a very simple graph 00:06:33.05 that gives you a sense 00:06:35.03 that we can turnover 00:06:36.26 this entire assembly line 00:06:38.27 in a test tube, 00:06:40.18 with a rate constant 00:06:42.15 that's approximately about 1/min. 00:06:47.10 So, approximately once every minute 00:06:49.17 this assembly line is releasing 00:06:51.21 6-Deoxyerythronolide B 00:06:53.22 in a test tube that is presented with the appropriate precursors, 00:06:57.27 and that is roughly the rate 00:06:59.24 we might expect this assembly line 00:07:01.18 to be working at 00:07:03.14 inside a cell. 00:07:05.06 I also wanna point out, 00:07:06.24 as the inset shows, 00:07:08.21 this assay is remarkably efficient. 00:07:12.29 Every equivalent of 6-Deoxyerythronolide B 00:07:16.28 has stoichiometric mapping 00:07:19.17 to an equivalent 00:07:21.26 of the propionyl-CoA primer 00:07:24.01 that is used, 00:07:25.28 and uses six equivalents of NADPH, 00:07:29.08 whose consumption is being measured 00:07:31.05 in this simple spectrophotometric assay. 00:07:35.07 Okay, so we have to tools to be able to study 00:07:39.04 the entire assembly line 00:07:40.25 inside a recombinant E. coli-like cell. 00:07:44.06 We have the ability 00:07:46.00 to study the entire assembly line 00:07:48.01 in a purified, reconstituted form. 00:07:51.03 We also have the ability, today, 00:07:53.21 to study the individual steps 00:07:56.12 in the catalytic cycle of these modules 00:08:01.09 in isolation. 00:08:03.10 So, recall in the previous module, 00:08:05.29 I introduced you to some of the 00:08:08.12 core reactions 00:08:10.18 that occur at every module. 00:08:13.00 There's a reaction 00:08:14.19 that we call chain translocation, 00:08:16.24 where the chain moves 00:08:18.08 from the acyl carrier protein 00:08:20.04 in the upstream module 00:08:21.26 to the module that's receiving the chain, 00:08:25.09 and if we want to interrogate 00:08:27.07 just that reaction 00:08:29.15 for one module, 00:08:31.09 what we do is pull out that module 00:08:34.07 from the rest of the assembly line, 00:08:36.17 purify that to homogeneity, 00:08:39.23 present it with 00:08:43.01 a chemoenzymatically-derived acyl carrier protein 00:08:47.00 that has the growing polyketide chain substrate 00:08:51.01 bound to it, 00:08:53.03 and we put it into a test tube 00:08:55.19 so that the chain translocation event 00:08:58.13 -- the movement of that growing polyketide chain 00:09:01.23 into the module -- 00:09:03.26 is the slow kinetic step, 00:09:06.00 and everything after that 00:09:08.03 that leads to the turnover of this module 00:09:10.21 is fast. 00:09:12.08 And so you can use 00:09:14.07 this kind of an assay 00:09:15.28 to interrogate, 00:09:18.01 using established kinetic paradigms, 00:09:20.23 that chain translocation step 00:09:23.10 of your favorite module 00:09:25.12 that you're interested in. 00:09:27.22 The same approach can also be used 00:09:31.03 to kinetically isolate the chain elongation event 00:09:34.11 that I introduced you to 00:09:36.03 in the earlier lecture. 00:09:38.18 So when we want to study chain elongation, 00:09:42.03 what we do is we take the module 00:09:44.22 whose elongation biochemistry 00:09:46.19 we want to study, 00:09:48.19 and we prepare just the ketosynthase 00:09:51.03 together with the acyltransferase 00:09:54.08 from that module 00:09:56.02 as one protein. 00:09:57.21 We produce its carrier protein, 00:10:00.04 its acyl carrier protein, 00:10:01.29 as another protein. 00:10:03.27 We put these two proteins together, 00:10:06.21 we present the two substrates 00:10:09.24 into this assay, 00:10:12.05 and we look for chain elongation, 00:10:15.00 which gives rise to the product. 00:10:17.19 And we do this under conditions 00:10:20.06 where the step we wanna probe, 00:10:22.07 the elongation step, 00:10:23.27 is the slow step, and everything else is fast. 00:10:27.20 You can do exactly the same thing 00:10:29.21 to probe the acyl transfer, 00:10:31.29 the selection of that building block 00:10:34.16 -- methylmalonyl coenzyme A-derived building block 00:10:38.12 from metabolism -- 00:10:40.08 the same approach can also work over there. 00:10:42.20 And all of these assays are well-developed, 00:10:44.29 they're in the literature, 00:10:46.19 and you can use them to study 00:10:48.08 your favorite assembly line. 00:10:51.07 In addition to those core reactions 00:10:53.28 -- chain translocation, 00:10:55.21 chain elongation, 00:10:57.08 and acyl transfer -- 00:10:58.23 I mentioned there are auxiliary reactions, 00:11:01.13 which I lumped under chain modification. 00:11:04.27 Those reactions include 00:11:06.22 ketoreductase-types of chemistries. 00:11:10.11 In this particular assay, 00:11:12.11 I'm adding the ketoreductase, 00:11:15.03 or KR, 00:11:16.26 as a stand-alone protein, 00:11:18.16 to the rest of my system 00:11:21.04 so that I can control the rate 00:11:22.27 at which that step occurs, 00:11:24.27 and I can look at the consequences 00:11:27.13 of putting one ketoreductase in my assay 00:11:30.04 as opposed to some other ketoreductase. 00:11:33.04 And that allows me to interrogate 00:11:35.28 the ketoreductase reaction. 00:11:38.00 You can do the same thing 00:11:40.04 at the level of the dehydratase reaction, 00:11:43.15 which follows after the ketoreductase reaction 00:11:47.29 in certain chain modification sequences. 00:11:52.00 So, all of these assays are also set up. 00:11:54.22 The point you need to recognize is that 00:11:57.13 you can probe through, again, 00:11:59.04 a divide-and-conquer approach, 00:12:01.00 the chemistry happening at any one of these steps 00:12:05.05 in the overall assembly line. 00:12:08.22 Using this combination of in vivo and in vitro tools, 00:12:14.02 there are a number of important problems 00:12:16.15 you can study. 00:12:17.28 In the remainder of this second module lecture, 00:12:21.16 I will talk to you about some examples 00:12:24.13 of questions, long-standing questions 00:12:26.26 in the field, 00:12:28.04 having to do with the specificity 00:12:29.28 of these assembly lines. 00:12:31.20 I'll give you two examples of those problems 00:12:33.22 because they have engineering implications. 00:12:36.21 And then in the next lecture we'll talk about 00:12:40.08 the assembly line mechanisms. 00:12:42.27 So, stereospecificity 00:12:46.01 is probably one of the most fascinating features 00:12:50.15 of these complex polyketide antibiotics 00:12:53.11 that these assembly lines make. 00:12:55.24 So, to the right, 00:12:57.14 you're seeing the 6-Deoxyerythronolide B product 00:13:01.16 of DEBS, 00:13:03.07 and for those of you who are looking at that now, 00:13:05.22 you're noticing that it has 10 stereocenters. 00:13:12.02 That is 2^10 possible chiral forms 00:13:17.08 of the same chemical formula, 00:13:20.01 or slightly more than 1000 00:13:22.12 of these chiral forms. 00:13:24.20 If you go to a fermentation plant 00:13:27.10 that makes erythromycin, 00:13:30.08 the large vat that produces erythromycin 00:13:33.23 has one out of those 1000+ 00:13:38.04 stereochemical forms in it; 00:13:40.17 the one that I'm showing you. 00:13:43.00 I think most of you would recognize 00:13:45.01 that that is a really impressive feat 00:13:48.04 on the part of nature... 00:13:49.27 how it can program this assembly line 00:13:52.06 to give one, and only one 00:13:54.13 stereochemical outcome. 00:13:56.22 That is a problem 00:13:58.29 that we have quite a good understanding of 00:14:02.16 how that happens today. 00:14:05.08 I've cited some references on this slide, 00:14:09.04 and so I will summarize for you 00:14:11.07 what these references teach us 00:14:13.18 about how stereochemistry is controlled 00:14:17.07 by the DEBS assembly line. 00:14:20.00 So, of those 10 stereocenters, 00:14:25.00 one of them, 00:14:27.12 which is this stereocenter, 00:14:31.06 is generated by 00:14:34.28 this ketoreductase 00:14:37.28 in Module 3 of the assembly line, 00:14:40.19 that I introduced to you as that epimerase, 00:14:44.29 that looks like a ketoreductase 00:14:46.29 in my previous lecture. 00:14:49.06 This is the enzyme that is a homologue 00:14:52.02 of other ketoreductases, 00:14:53.18 but does no NADPH-dependent chemistry. 00:14:57.27 Instead, it epimerizes the C2 carbon atom 00:15:01.20 of the growing polyketide chain 00:15:04.28 that is lodged in Module 3 00:15:06.25 of the assembly line. 00:15:09.05 Of the remaining 9 stereocenters, 00:15:12.07 8 of those stereocenters 00:15:14.22 are shown in red, 00:15:17.09 and they are controlled 00:15:19.21 by the 3 red ketoreductases 00:15:23.02 and 1 blue ketoreductase 00:15:25.17 in Modules 1, 2, 5, and 6, respectively. 00:15:32.23 So, each of these 4 ketoreductases 00:15:37.25 controls 2 stereocenters apiece. 00:15:42.20 For the chemically initiated, 00:15:44.29 these enzymes are not just stereoselective, 00:15:48.22 they're also diastereoselective; 00:15:51.17 so they're setting 2 stereocenters at a time. 00:15:56.01 And these enzymes, we know... 00:15:58.23 these 4 enzymes we know, today, 00:16:01.13 are both necessary and sufficient 00:16:04.10 for the unique labeling... 00:16:08.05 for the unique identification 00:16:11.01 of those stereocenters. 00:16:13.21 The last stereocenter, 00:16:16.01 which is this stereocenter, 00:16:19.13 is at the 6 position, 00:16:22.01 is a more complex output, 00:16:25.08 and it is generated by 3 enzymes 00:16:27.28 in Module 4 of DEBS. 00:16:30.28 There is a ketoreductase, 00:16:34.03 a dehydratase, 00:16:36.04 and an enoylreductase, 00:16:38.08 that all collaborate with each other 00:16:41.06 to set this one stereocenter. 00:16:47.06 In addition to stereochemistry, 00:16:49.20 there is another very important 00:16:53.15 specificity that is encoded 00:16:56.11 in this assembly line 00:16:58.22 at each module, 00:17:00.24 and that is the specificity 00:17:05.17 that corresponds to the choice 00:17:08.19 of the extender unit. 00:17:10.19 In my introduction, 00:17:12.07 I pointed out that all of the modules 00:17:14.26 of 6-Deoxyerythronolide B synthase 00:17:18.04 use a methylmalonyl coenzyme A 00:17:21.14 extender unit. 00:17:23.22 In the case of DEBS, 00:17:27.06 the R group that is shown 00:17:29.25 in this enzymatic scheme 00:17:32.07 would be a methyl group. 00:17:34.17 Other polyketide synthases 00:17:37.06 can use coenzyme A thioesters 00:17:39.29 that contain other functional groups 00:17:42.16 in place of a methyl group, over here. 00:17:46.11 And all of these choices 00:17:48.18 are made by the acyltransferase. 00:17:53.00 These acyltransferases 00:17:55.10 are relatively specific. 00:17:58.01 Not only do they have high specificity, 00:18:01.04 as shown in this graph 00:18:03.15 for the coenzyme A precursor 00:18:05.28 they're picking from the metabolic soup... 00:18:09.09 so what you see in this graph over here 00:18:13.06 is the rate... 00:18:15.24 the velocity versus substrate concentration 00:18:18.20 of the preferred substrate, 00:18:20.20 which is methylmalonyl coenzyme A, 00:18:23.22 and down here are the rates 00:18:25.25 if R is one methyl short, 00:18:28.13 so in other words it's a hydrogen instead of a methyl, 00:18:31.14 or one methyl longer, 00:18:33.23 which is an ethyl group. 00:18:35.29 And as you can tell, 00:18:37.25 this acyltransferase 00:18:39.18 that we're showing you data for in this slide 00:18:42.08 is highly selective 00:18:44.24 for a methyl group 00:18:46.25 instead of one smaller or one larger. 00:18:50.16 Now, in addition to being specific 00:18:52.25 for its cognate substrate, 00:18:56.09 this enzyme is also specific 00:18:59.11 for its protein partner, 00:19:01.09 which is the acyl carrier protein, 00:19:03.22 that is being used. 00:19:05.25 And here I'm introducing you 00:19:07.25 to a concept that is gonna come back 00:19:10.11 in a more significant way 00:19:12.13 in the last of my three lectures, 00:19:14.16 which is the importance 00:19:16.10 of protein-protein interactions 00:19:19.04 in the assembly line biochemistry 00:19:22.00 of these systems. 00:19:23.22 In this case, what you're seeing is 00:19:27.03 that the acyl carrier protein 00:19:29.21 is being strongly recognized 00:19:32.00 by the acyltransferase, 00:19:34.05 because if you give this same acyltransferase 00:19:37.18 other acyl carrier proteins 00:19:39.25 from other modules of DEBS 00:19:41.23 or elsewhere, 00:19:43.19 they work much more poorly 00:19:46.09 than the natural acyl carrier protein. 00:19:49.19 So in addition to recognizing 00:19:51.15 the coenzyme A precursor, 00:19:53.27 you also have recognition 00:19:56.00 of the acyl carrier protein. 00:19:58.18 Now, for those of you who are familiar 00:20:00.16 with enzymes kinetics 00:20:02.16 know that from data like this, 00:20:04.18 you can derive mechanisms 00:20:06.10 of how these enzymes work. 00:20:08.15 So, in this case, 00:20:10.19 this acyltransferase 00:20:13.02 has a ping-pong 00:20:15.03 bi-bi-type of a mechanism. 00:20:18.11 The coenzyme A precursor first comes in, 00:20:21.21 it is bound by the acyltransferase, 00:20:25.05 the acyltransferase picks the methylmalonyl extender unit, 00:20:29.02 coenzyme A leaves, 00:20:30.23 the carrier protein comes in, 00:20:32.29 is recognized by the acyltransferase, 00:20:35.25 and takes away the product 00:20:38.06 - the methylmalonyl extender unit. 00:20:40.23 So that ping-pong element comes into 00:20:43.12 this kind of a mechanism. 00:20:45.12 Now, you can also ask, 00:20:46.29 in addition to these gatekeeper acyltransferases 00:20:50.25 that control the choice of the building block, 00:20:55.18 there are many other enzymes 00:20:57.18 in this assembly line 00:20:59.17 that lie downstream of each choice 00:21:02.15 that a module makes 00:21:04.16 of its building block. 00:21:06.09 To what extent do they influence 00:21:09.05 the overall substrate specificity? 00:21:11.29 Do they care about what the upstream module chose 00:21:16.17 as its precursor for elongating 00:21:20.01 the growing polyketide chain? 00:21:22.13 We can ask questions like that 00:21:24.16 using the assays... biochemical assays 00:21:27.11 I showed you earlier on, 00:21:29.16 and from those experiments you learn something 00:21:31.22 quite interesting. 00:21:33.16 So, you learn that the downstream steps, 00:21:36.14 beyond the acyltransfer step, 00:21:38.28 in many modules 00:21:41.12 are not that discriminatory, 00:21:43.09 analogous to what Henry Ford 00:21:45.11 had contemplated for his assembly line. 00:21:48.26 The downstream steps 00:21:51.01 have low, but not a significant amount, 00:21:54.16 of specificity. 00:21:56.21 So if, by whatever mechanism, 00:21:59.14 you can fool this acyltransferase 00:22:02.25 to put a hydrogen instead of a methyl 00:22:06.16 at this position on the carrier protein, 00:22:09.26 the elongation enzyme 00:22:12.06 that elongates the chain 00:22:14.10 and puts this R group in the growing polyketide chain, 00:22:19.14 primarily loses 00:22:22.12 about 2- to 4-fold specificity 00:22:25.10 as a result of this mistake 00:22:28.13 that the upstream step made. 00:22:31.00 That's not much in the grand scheme of things, 00:22:33.25 but what you have to remember is 00:22:37.02 these assembly lines have 00:22:39.18 many, many downstream steps. 00:22:41.27 So, one of these assembly lines, 00:22:43.23 the first module over here, or the second module, 00:22:46.28 has four or five modules downstream 00:22:50.17 that are looking at the consequences 00:22:53.01 of what that module did. 00:22:55.00 And so these small effects 00:22:57.09 at the level of substrate specificity 00:22:59.23 then have quite significant impact 00:23:02.21 on the final product, 00:23:04.18 and you can see this 00:23:06.18 in the context of assays like this. 00:23:09.06 So here, 00:23:10.24 in the spirit of engineering, 00:23:12.17 what we're doing is we're taking that natural... 00:23:15.17 the natural assembly line that nature uses 00:23:18.04 to make 6-Deoxyerythronolide B, 00:23:20.27 we're taking that full assembly line, 00:23:23.00 and instead of just presenting it 00:23:25.27 methylmalonyl coenzyme A, 00:23:28.14 we're now presenting it a 1:1 mixture 00:23:32.06 of methylmalonyl coenzyme A 00:23:34.11 and ethylmalonyl coenzyme A, 00:23:36.28 and we're asking what's gonna happen. 00:23:40.11 Are you gonna get just 6-Deoxyerythronolide B? 00:23:44.19 Are you gonna get something else, 00:23:47.10 one or two other products? 00:23:49.06 Or are you gonna get a zoo of products? 00:23:51.26 And the answer to that is, 00:23:53.28 you get some analogues 00:23:56.23 that are produced competitively 00:23:58.25 with the natural product 6-DEB, 00:24:02.23 but these compounds 00:24:05.24 aren't immediately obvious 00:24:08.00 why these should be formed 00:24:10.02 and other ones shouldn't be formed. 00:24:11.29 So at least one of these, for example, 00:24:13.27 this peak that I show you out here, 00:24:16.04 whose mass spectrum is shown over here, 00:24:18.22 we know with reasonable confidence, 00:24:21.06 has an ethyl group 00:24:23.07 that is incorporated at the C8 position 00:24:26.12 instead of a methyl group. 00:24:28.06 So, somehow, 00:24:30.10 that gatekeeper transferase 00:24:32.29 has enough tolerance 00:24:35.04 for an ethylmalonyl extender unit, 00:24:37.24 and all of the downstream enzymes 00:24:40.13 on the assembly line 00:24:42.11 are sufficiently tolerant 00:24:44.13 that they will let that mistake slide by, 00:24:46.28 so you get this desired product. 00:24:50.05 And if we could predict 00:24:52.10 what's gonna be made and what's not gonna be made 00:24:55.01 through an experiment like this, 00:24:57.05 why, then, we would have a way to precisely engineer 00:24:59.21 an antibiotic like erythromycin 00:25:01.21 to make a molecule like this. 00:25:03.27 But right now, we're just beginning to scratch 00:25:06.10 the tip of the iceberg 00:25:07.22 in terms of what's possible, 00:25:09.13 and what's not, 00:25:11.04 in these kinds of systems, 00:25:12.21 and an experiment like this illustrates 00:25:14.22 what's possible. 00:25:16.28 These kinds of insights can also be used 00:25:19.12 for most sophisticated engineering experiments, 00:25:22.07 analogous to the kinds of experiments 00:25:24.17 you may be familiar with 00:25:26.25 when you think about incorporating unnatural amino acids 00:25:31.18 in proteins that are derived 00:25:33.24 by ribosomal mechanisms. 00:25:36.04 And so, in this, 00:25:38.08 there are some examples of acyltransferases 00:25:42.10 that are what we call stand-alone acyltransferases. 00:25:46.12 They operate outside the assembly line, 00:25:49.21 and because they work 00:25:51.29 very, very fast compared to typical assembly line acyltransferases 00:25:58.03 that exist in assembly lines, 00:26:00.16 you can do some interesting experiments. 00:26:02.25 So in this case, 00:26:04.24 we have knocked out 00:26:07.10 one acyltransferase 00:26:09.17 out of all the six acyltransferases 00:26:12.09 on the 6-Deoxyerythronolide B synthase 00:26:16.23 -- so this is like a site-directed mutant 00:26:19.14 that has inactivated that acyltransferase -- 00:26:23.02 and we complement it, 00:26:25.15 in trans, 00:26:27.26 with that very ultra-fast acyltransferase 00:26:30.27 that we get from a different assembly line 00:26:34.02 in nature. 00:26:36.07 And what happens is, 00:26:38.00 because this acyltransferase 00:26:40.09 will pick a malonyl unit instead of a methylmalonyl unit, 00:26:44.13 it can transfer that malonyl unit 00:26:47.07 onto this module, 00:26:49.02 because this module is simply waiting 00:26:50.26 for something to come its way, 00:26:52.29 and this enzyme can do it pretty quickly, 00:26:56.18 and now that resulting intermediate 00:26:58.27 moves all the way down in the assembly line 00:27:01.23 to give you a product 00:27:04.09 that has one and only one change 00:27:07.11 in the entire macrocycle 00:27:10.09 that is made. 00:27:12.08 And what you see over here is, 00:27:13.29 if I have my assembly line that is present 00:27:18.15 at say micromolar concentrations in my assay, 00:27:22.00 at nanomolar concentrations 00:27:24.26 I can get quite respectable incorporation 00:27:29.22 of malonyl coenzyme A 00:27:31.18 at that single position, 00:27:33.16 to give me 12-desmethyl-6-Deoxyerythronolide B. 00:27:39.25 So this is a way you can cheat the system, 00:27:43.26 so long as you have 00:27:46.19 a robust enzyme 00:27:49.00 that can trans-complement the module 00:27:51.15 that you wanna cheat. 00:27:53.22 So hopefully that gives you 00:27:55.24 a flavor of the kinds of tools we have 00:27:58.28 and the way we use these tools 00:28:01.01 to study specificity. 00:28:03.12 Thank you.