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5.13E: Polyketide Antibiotics - Biology

5.13E: Polyketide Antibiotics - Biology


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Polyketides are secondary metabolites produced from bacteria, fungi, plants, and animals.

Learning Objectives

  • Describe the characteristics associated with polyketides, including: type I, II and III polyketides

Key Points

  • Secondary metabolites are organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism.
  • Polyketides are usually biosynthesized through the decarboxylative condensation of malonyl-CoA derived extender units in a similar process to fatty acid biosynthesis.
  • Polyketides are structurally a very diverse family of natural products with diverse biological activities and pharmacological properties.

Key Terms

  • Polyketides: Polyketides are secondary metabolites from bacteria, fungi, plants, and animals. Polyketides are usually biosynthesized through the decarboxylative condensation of malonyl-CoA derived extender units in a similar process to fatty acid synthesis (a Claisen condensation).
  • metabolites: Metabolites are the intermediates and products of metabolism. The term metabolite is usually restricted to small molecules. Metabolites have various functions, including fuel, structure, signaling, stimulatory and inhibitory effects on enzymes, catalytic activity of their own (usually as a cofactor to an enzyme), defense, and interactions with other organisms (e.g. pigments, odorants, and pheromones).
  • biosynthesized: Biosynthesis (also called biogenesis or “anabolism”) is an enzyme-catalyzed process in cells of living organisms by which substrates are converted to more complex products. The biosynthesis process often consists of several enzymatic steps in which the product of one step is used as substrate in the following step.

Polyketides are secondary metabolites produced from bacteria, fungi, plants, and animals.

Secondary metabolites are organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism. Unlike primary metabolites, the absence of secondary metabolites does not result in immediate death, but rather in long-term impairment of the organism’s survivability, fecundity, or aesthetics, or perhaps in no significant change at all. Secondary metabolites are often restricted to a narrow set of species within a phylogenetic group. Secondary metabolites often play an important role in plant defense against herbivory and other interspecies defenses. Humans use secondary metabolites as medicines, flavorings, and recreational drugs.

Polyketides are usually biosynthesized through the decarboxylative condensation of malonyl-CoA derived extender units in a similar process to fatty acid biosynthesis (a Claisen condensation). The polyketide chains produced by a minimal polyketide synthase are often further derivitized and modified into bioactive natural products.

Polyketides are structurally a very diverse family of natural products with diverse biological activities and pharmacological properties. They are broadly divided into three classes: type I polyketides (often macrolides produced by multimodular megasynthases), type II polyketides (often aromatic molecules produced by the iterative action of dissociated enzymes ), and type III polyketides (often small aromatic molecules produced by fungal species). Polyketide antibiotics, antifungals, cytostatics, anticholesteremic, antiparasitics, coccidiostats, animal growth promoters, and natural insecticides are in commercial use.

Examples of polyketides include: Macrolides; Pikromycin, the first isolated macrolide; the antibiotics erythromycin A; clarithromycin, and azithromycin; the immunosuppressant tacrolimus; Radicicol and Pochonin family (HSP90 inhibitor); Polyene antibiotics; Amphotericin; Tetracyclines and the tetracycline family of antibiotics.

Polyketides are synthesized by one or more specialized and highly complex polyketide synthase (PKS) enzymes.


Fungal Secondary Metabolism

Francesco Vinale , . Santiago Gutiérrez , in Reference Module in Life Sciences , 2021

Polyketides

Polyketides are the amplest class of fungal SMs and are biosynthesized by type I polyketide synthases (PKSs). Short-chain carboxylic acids, typically acetyl-coenzyme A (acetyl-CoA) and malonyl-CoA, are condensed to form carbon chains of variable lengths. The main difference between polyketides and fatty acids is the full reduction of the β-carbon in fatty acids, which is an optional event in polyketide synthesis. The variety of fungal polyketide chemical structures is a consequence of many iteration reactions, the number of reduction reactions, which extender unit is used and, in the case of aromatic polyketides, cyclization of the nascent polyketide chain. Other structures are related to the introduction of diverse post-polyketide-synthesis steps.


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.


The Streptomyces glaucescens tcmKL polyketide synthase and tcmN polyketide cyclase genes govern the size and shape of aromatic polyketides

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Antibiotics Types: Top 7 Types of Antibiotics (With Diagram)

This article throws light upon the top seven types of antibiotics. The seven types of antibiotics are: (1) Penicillins (2) Cephalosporin’s (3) Aminoglycosides (4) Tetracyclines (5) Macrolides (6) Aromatic Antibiotics and (7) Nucleoside Antibiotics.

Type # 1. Penicillins:

Penicillins are a group of β-lactam containing bactericidal antibiotics. Being the first among the antibiotics to be discovered, penicillins are historically important. The structures of important synthetic and semi-synthetic penicillins are depicted in Fig. 25.1. The basic structure of all the penicillins consists of a lactam ring and a thizolidine ring fused together to form 6-aminopenicillanic acid.

Action of Penicillins:

Natural penicillins (penicillins V and G) are effective against several Cram-positive bacteria. They inhibit the bacterial cell wall (i.e. peptoglycan) synthesis and cause cell death. Some persons (approximately 0.5-2% of population) are allergic to penicillin.

Natural penicillins are ineffective against microorganisms that produce β-lactamase, since this enzyme can hydrolyse penicillins e.g. Staphylococcus aureus. Several semi-synthetic penicillins that are resistant to β-lactamase have been developed and successfully used against a large number of Gram-negative bacteria.

Cloxacillin, ampicillin, floxacillin and azlocillin are some examples of semi-synthetic penicillins. These are quite comparable in action to cephalosporin’s. From the huge quantities of penicillins produced by fermentation, about 40% are used for human healthcare, 15% for animal healthcare and 45% for the preparation of semi-synthetic penicillins.

Organisms for Penicillin Production:

In the early days, Penlcillium notatum was used for the large-scale production of penicillins. Currently, Penicillium chrysogenum and its improved mutant strains are preferred. Previously, the penicillin production used to be less than 2 units/ml, and with the new strains, the production runs into several thousands of units/ml. One of the high yielding strains wis Q176 is preferred by several penicillin manufacturers.

Genetic engineering for improved penicillin production:

Some of the genes involved in penicillin biosynthesis by P. chrysogenum have been identified. Genetic manipulations were carried out so as to substantially increase the penicillin production. For instance, extra genes coding for the enzymes cyclase and acyltransferase have been inserted into C. chrysogenum.

Biosynthesis of Penicillin:

L-α-Aminoadipic acid combines with L-cysteine, and then with L-valine to form a tripeptide namely α-L-aminoadipylcysteinylvaline. This compound undergoes cyclization to form isopenicillin which reacts with phenyl acetyl CoA (catalysed by the enzyme acyltransferase) to produce penicillin G (benzyl penicillin). In this reaction, aminoadipic acid gets exchanged with phenylacetic acid (Fig. 25.2).

Regulation of biosynthesis:

Some of the biochemical reactions for the synthesis of penicillin and lysine are common. Thus, L-α-aminoadipic acid is a common intermediate for the synthesis of penicillin and lysine. The availability of aminoadipic acid plays a significant role in regulating the synthesis of penicillin.

Penicillin biosynthesis is inhibited by glucose through catabolite repression. For this reason, penicillin was produced by a slowly degraded sugar like lactose. The concentrations of phosphate and ammonia also influence penicillin synthesis.

Production Process of Penicillin:

An outline of the flow chart for the industrial production of penicillin is depicted in Fig. 25.3. The lyophilized culture of spores is cultivated for inoculum development which is transferred to pre-fermenter, and then to fermenter.

Penicillin production is an aerobic process and therefore, a continuous supply of O2 to the growing culture is very essential. The required aeration rate is 0.5-1.0 vvm. The pH is maintained around 6.5, and the optimal temperature is in the range of 25-27°C. Penicillin production is usually carried out by submerged processes. The medium used for fermentation consists of corn steep liquor (4-5% dry weight) and carbon source (usually lactose). An addition of yeast extract, soy meal or whey is done for a good supply of nitrogen.

Sometimes, ammonium sulfate is added for the supply of nitrogen. Phenylacetic acid (or phenoxyacetic acid) which serves as a precursor for penicillin biosynthesis is continuously fed. Further, continuous feeding of sugar is advantageous for a good yield of penicillin. The penicillin production profiles are depicted in Figs. 25.4 and Fig. 25.5.

It is estimated that approximately 10% of the metabolised carbon contributes to penicillin production, while 65% is utilised towards energy supply and 25% for growth of the organisms. The efficiency of penicillin production can be optimized by adequate supply of carbon source. Thus, by adding glucose and acetic acid, the yield can be increased by about 25%.

For efficient synthesis of penicillin, the growth of the organism from spores must be in a loose form and not as pellets. The growth phase is around 40 hours with a doubling time of 6-8 hours. After the growth phase is stabilized, the penicillin production exponentially increases with appropriate culture conditions. The penicillin production phase can be extended to 150-180 hours.

Recovery of Penicillin:

As the fermentation is complete, the broth containing about 1% penicillin is processed for extraction. The mycelium is removed by filtration. Penicillin is recovered by solvent (n-butyl acetate or methyl ketone) extraction at low temperature (<10°C) and acidic pH (<3.0). By this way, the chemical and enzymatic (bacterial penicillinase) degradations of penicillin can be minimized.

The penicillin containing solvent is treated with activated carbon to remove impurities and pigments. Penicillin can be recovered by adding potassium or sodium acetate. The potassium or sodium salts of penicillin can be further processed (in dry solvents such as n-butanol or isopropanol) to remove impurities. The yield of penicillin is around 90%.

As the water is totally removed, penicillin salts can be crystallized and dried under required pressure. This can be then processed to finally produce the pharmaceutical dosage forms. Penicillins G and H are the fermented products obtained from the fungus Penicillium chrysogenum.

Production of 6-Amino Penicillanic Acid:

The penicillins G and H are mostly used as the starting materials for the production of several synthetic penicillins containing the basic nucleus namely 6-amino penicillanic acid (6-APA). About 10 years ago, only chemical methods were available for hydrolysis of penicillins to produce 6-APA. Now a days, enzymatic methods are preferred.

Immobilized penicillin amidases enzymes have been developed for specific hydrolysis of penicillin G and penicillin V. Penicillin salt of either G or V can be used for hydrolysis by immobilized enzyme system. The pH during hydrolysis is kept around 7-8, and the product 6-APA can be recovered by bringing down the pH to 4.

At pH 4, 6-amino penicillanic acid gets precipitated almost completely in the presence of a water immiscible solvent. In general, the enzymatic hydrolysis is more efficient for penicillin V than for penicillin G. However, penicillin G is a more versatile compound, as it is required for ring expansions.

Type # 2. Cephalosporin’s:

The pharmaceutical uses of penicillins are associated with allergic reactions in some individuals. To overcome these allergic problems, cephalosporin’s were developed. They have improved stability against β-lactamases, and are more active against Gram-negative bacteria. Cephalosporin’s are broad spectrum antibiotics with low toxicity. The structures of different cephalosporin’s are shown in Fig. 25.6. Basically cephalosporin’s have a β-lactam ring fused with a dihydrothiazine ring.

Organisms for Cephalosporin Production:

Cephalosporin C was first discovered in the cultures of fungus Cephalosporium acremonium (later renamed as Acremonium chreysogenum) and this organism continuous to be used even today. The other organisms employed for cephalosporin production are Emericeliopsis sp, Paecilomyces sp and Streptomyces sp.

Several mutants of C. acremonium have been developed for improved production of cephalosporin. Mutants with defective sulfur metabolism or those with resistance to sulfur analogs have high yielding capacity. Certain regulatory genes of cephalosporin biosynthesis (e.g., isopenicillin N synthetase) have been cloned and genetic manipulations carried out for increased production of cephalosporin’s.

Biosynthesis of Cephalosporin:

The early stages of the biosynthetic pathway for cephalosporin are the same as that for penicillin synthesis (See Fig. 25.2). As the tripeptide (aminoadipylcysteinylvaline) is formed, it undergoes cyclization to produce isopenicillin N. By the action of epimerase, penicillin N is formed from isopenicillin N. Then, penicillin N gets converted to cephalosporin C by a three stage reaction catalysed by three distinct enzymes namely expandase, hydroxylase and acetyl transferase (Fig. 25.7).

Regulation of biosynthesis:

A low concentration of lysine promotes cephalosporin synthesis. The inhibitory effect of lysine at a higher concentration can be overcome by adding L-aminoadipic acid. The carbon sources that get rapidly degraded (e.g. glucose, glycerol) reduce cephalosporin production. Methionine promotes cephalosporin synthesis in C. acremonium, but has no influence on Streptomyces’s.

Production Process of Cephalosporin:

The fermentation process concerned with the production of cephalosporin is similar to that of penicillin. The culture media consists of corn steep liquor and soy flour-based media in a continuous feeding system. The other ingredients of the medium include sucrose, glucose and ammonium salts. Methionine is added as a source of sulfur.

The fermentation is carried out at temperature 25-28°C and pH 6-7. The growth of micro­organisms substantially increases with good O2 supply, although during production phase, O2 consumption declines. Cephalosporin C from the culture broth can be recovered by ion-exchange resins, and by using column chromatography. Cephalosporin C can be precipitated as zinc, sodium or potassium salt, and isolated.

Chemical synthesis of cephalosporin:

In recent years, by using penicillin V as the starting material, chemical synthesis of cephalosporin has become possible. This is being done due to low cost of production of penicillin.

Production of 7-Aminocephalosporanic Acid:

7-Aminocephalosporonic acid (7-ACA) is the nucleus structure present in all the cephalosporin’s. Cephalosporin C, produced by fermentation, can be subjected to chemical hydrolysis to form 7-ACA. This is tedious, and is associated with several drawbacks.

Recently, enzymatic hydrolysis of cephalosporin C to 7-ACA has been developed. This is mainly carried out by two enzymes-D-amino acid oxidase (isolated from Trigonopsis variabilis) and glutaryl amidase (source-Pseudomonas sp). Bio- technologists have been successful in immobilizing these two enzymes for efficient and large scale manufacture of 7-ACA.

New β-Lactam Technology for Production of 7-ACA:

Scientists have been successful in producing 7-aminocephalosporanic acid by P. chrysogenum fermentation. This is possible through genetic manipulations. As already described in cephalosporin biosynthesis (Fig. 25.7), penicillin N is the substrate for the enzyme expandase. Adipyl- 6-aminopenicillanic acid (produced by P. chrysogenum on adding adipic acid) which resembles in structure with penicillin N, can also serve as a substrate for expandase.

By inserting expandase and hydroxylase gene (cefEF), and acetyl transferase gene (cefG) from S. clavuligerus into P. chrysogenum, the production of adipyl-7-ACA has become possible (Fig. 25.8). Further, the genes responsible for the enzymes D-amino acid oxidase (from Pseudomonas diminuta) have also been inserted into P. chrysogenum. Both these enzymes act on adipyl-7-ACA to produce 7-amino- cephalosporanic acid.

Type # 3. Aminoglycosides:

Aminoglycosides are oligosaccharide (carbohy­drate) antibiotics. They contain an aminocyclo-hexanol moiety which is bound to other amino sugars by glycosidic linkages. More than 100 aminoglycosides are known e.g. streptomycin, neomycin, kanamycin, gentamicin, hygromycin, sisomicin.

Aminoglycosides are very potent antibiotics and act against Gram-positive and Gram-negative bacteria, besides mycobacteria. At the molecular level, aminoglycosides bind to 30S ribosome and block protein biosynthesis. Prolonged use of aminoglycosides causes damage to kidneys, and hearing impairment.

For the treatment of severe and chronic infections, aminoglycosides are the antibiotics of choice. Streptomycin was the first aminoglycoside that was successfully used to treat tuberculosis (i.e. against Mycobacterium tuberculosis). Usually, aminoglycosides are regarded as reserve antibiotics, since resistance may develop easily.

Organisms for Aminoglycoside Production:

Aminoglycoside antibiotics are produced by Actinomyces sp. Some examples are given in Table 25.2. Recombinant DNA techniques have been used to produce hybrid aminoglycosides, and for increasing the fermentation yield.

Biosynthesis of Aminoglycosides:

All the ring structures in the molecules of aminoglycosides are ultimately derived from glucose. Most of the biosynthetic pathways concerned with the formation of at least some aminoglycosides have been elucidated.

Biosynthesis of Streptomycin:

The outline of the pathway for the synthesis of streptomycin is depicted in Fig. 25.9. More than 30 enzymatic steps have been identified. Glucose 6-phosphate obtained from glucose takes three independent routes to respectively produce streptidine 6-phosphate, L-dehydrostreptose and N- methyl glucosamine.

The former two compounds condense to form an intermediate which later combines with methyl glucosamine to produce di-hydro-streptomycin-6-phosphate. This compound, in the next of couple of reactions, gets converted to streptomycin.

Regulation of biosynthesis:

Very little is known about the regulation of streptomycin synthesis. A compound named as factor A (chemically isocapryloyl-hydroxymethyl-γ-butyrate) has been isolated from streptomycin-producing strains of S. griseus. Factor A promotes streptomycin production. In fact, factor A- mutants that cannot synthesize streptomycin have been isolated. They can synthesize streptomycin on adding factor A. The nutrient sources-carbohydrates (glucose), ammonia and phosphate also regulate (by feedback mechanism) streptomycin production.

Production Process of Streptomycin:

The medium used for streptomycin usually consists of soy meal or soy flour or corn syrup that can supply glucose at a slow rate (amylase activity is poor in Streptomyces sp). The initial supply of nitrogen (NH3) and phosphate is also obtained from soy meal. This is required since glucose, ammonia and phosphate in high quantities inhibit streptomycin synthesis.

The fermentation conditions for optimal production of streptomycin are — temperature 27-30°C, pH 6.5-7.5, aeration rate 0.5-1.0 vvm. The duration of fermentation process depends on the strain used, and is between 6 to 8 days.

Recovery of Streptomycin:

Streptomycin or other aminoglycosides are basic in nature. They can be recovered by weak cationic exchange resins in an ion- exchange column. Treatment with activated carbon is often necessary to remove impurities. Streptomycin can be precipitated in the form of sulfate salt.

Type # 4. Tetracyclines:

Tetracyclines are broad spectrum antibiotics with widespread medical use. They are effective against Gram-positive and Gram-negative bacteria, besides other organisms (mycoplasmas, chlamydias rickettsias). Tetracyclines are used to combat stomach ulcers (against Helicobacter pylori). They are the most commonly used antibiotics, next to cephalosporin’s and penicillins. Tetracyclines inhibit protein biosynthesis by blocking the binding of aminoacyl tRNA to ribosomes (A site).

The basic structure of tetracyclines is composed of a naphthalene ring (a four ring structure). The substituent groups of the common tetracyclines are given in Fig. 25.10. Among these, chlortetracycline and oxy-tetracycline are most commonly used in the treatment of human and veterinary diseases, besides in the preservation of fish, meat and poultry (in some countries).

Organisms for Tetracycline Production:

The first tetracycline antibiotic that was isolated was chlortetracycline from the cultures of Streptomyces aureofaciens (in 1945). There are at least 20 streptomycetes identified now that usually produces a mixture of tetracyclines. In the Fig. 25.10, a selected list of these organisms for producing tetracyclines is also given.

High-yielding strains of S. aureofaciens and S. rimosus have been developed by using ultraviolet radiation and/or other mutagens (nitrosoguanidine). Such strains are very efficient for the production of chlortetracycline. Further, genetically engineered strains of S. rimosus have been developed for increased synthesis of oxytetracycline.

Biosynthesis of Tetracyclines:

The pathway for the biosynthesis of tetracyclines is very complex. An outline of the synthesis of chlortetracycline by S. aureofaciens is given in Fig. 25.11. There are at least 72 intermediates formed during the course of chlortetracycline biosynthesis, some of them have not been fully characterized.

Polyketide antibiotic synthesis:

The term polyketide refers to a group of antibiotics that are synthesized by successive condensation of small carboxylic acids such as acetate, butyrate, propionate and malonate. The synthesis of polyketide antibiotics is comparable to that of long chain fatty acids. That is the carbon chain grows by cyclic condensation process. The synthesis of tetracyclines is a good example of polyketide antibiotic synthesis.

As glucose gets oxidised, it forms acetyl CoA and then malonyl CoA. On transamination, the later gives malonomoyl CoA. The enzyme anthracene synthase complex binds to malonomoyl CoA and brings out the condensation of 8 molecules of malonyl CoA to form a polyketide intermediates (four ring structures). These intermediates undergo a series of reactions to finally produce chlortetracycline.

Regulation of biosynthesis:

Carbohydrate metabolism (particularly glycolysis) controls chlortetracycline synthesis. For more efficient synthesis of the antibiotic, glycolysis has to be substantially low. The addition of phosphate reduces chlortetracycline production.

Production Process of Chlortetracycline:

The fermentation medium consists of corn steep liquor, soy flour or peanut meal for the supply of nitrogen and carbon sources. Continuous feeding of carbohydrate is desirable for good growth of the organism and production of the antibiotic. This can be done either by addition of crude carbon sources or by supplying glucose or starch. For more efficient production of chlortetracycline, the supply of ammonium and phosphate has to be maintained at a low concentration.

An outline of the production process for chlortetracycline is depicted in Fig. 25.12. The ideal fermentation conditions are — temperature 27-30°C, pH-6.5-7.5, and aeration 0.8-1.0 vvm. The duration of fermentation is around 4 days.

Recovery of chlortetracycline:

At the end of the fermentation, the culture broth is filtered to remove the mycelium. The filtrate is treated with n-butanol or methylisobutylketone in acidic or alkaline condition for extracting the antibiotic. It is then absorbed to activated charcoal to remove other impurities. Chlortetracycline is eluted and crystallized.

Production of Tetracycline —Different Processes:

The production of tetracycline can be achieved by one or more of the following ways.

i. By chemical treatment of chlortetracycline.

ii. By carrying out fermentation in a chloride-free culture medium.

iii. By employing mutants in which chlorination reaction does not occur.

iv. By blocking chlorination reaction by the addition of inhibitors e.g. thiourea, 2-thiouracil.

Type # 5. Macrolides:

Macrolides are a group of antibiotics with large lactone rings (i.e. macrocylic lactone rings). They consist of 12-, 14-, or 16-membered lactone rings with 1-3 sugars linked by glycosidic bonds. The sugars may be 6-deoxyhexoses or amino sugars. Erythromycin and oleandomycin are 14-membered (lactone ring containing) macrolides while leucomycin and tylosin are examples for 16-mem­bered microlides.

Erythromycin and its derivative clarithromycin are the most commonly prescribed microlides. They are effective against Gram-positive bacteria, and are frequently used to kill penicillin-resistant organisms. Clarithromycin is currently used to combat stomach ulcers caused by H. pylori. The macrolides inhibit the protein biosynthesis by binding to 50S ribosome. Polyene macrolides is the term applied for very large ring macrolides that many contain lactone rings in the range of 26-28. e.g. nystatin, amphotericin. These polyene macrolides are antifungal.

Production of Macrolides:

Macrolides are produced by actinomycetes. The major macrolide antibiotics and the corresponding organisms synthesizing them are given in Table 25.3.

Biosynthesis of Erythromycin:

In the biosynthesis of erythromycin, the lactone rings are contributed by acetate, propionate or butyrate while the sugar units are derived from glucose. Macrolide biosynthesis is a complex process and a good example of polyketide synthesis which is analogous to fatty acid biosynthesis. The enzyme lactone synthase is a multi-enzyme complex which is comparable in its structure and function to fatty acid synthase complex. An outline of the biosynthesis of erythromycin is given in Fig. 25.13.

Regulation of biosynthesis:

End product inhibition of erythromycin synthesis is well documented. Erythronolide B inhibits the enzyme lactone synthase. The final product erythromycin has also been shown to inhibit certain enzymes of the pathway (e.g. transmethylase). Addition of propanol to the culture medium induces the synthesis of acetyl CoA carboxylase, and almost doubles the production of erythromycin.

Production Process of Erythromycin:

Industrial production of erythromycin is carried out by aerobic submerged fermentation. The culture medium mainly consists of soy meal or corn steep liquor, glucose (or starch), yeast extract and ammonium sulfate. Fermentation is carried out at 30-34°C for about 3-7 days. Conventional methods are used for the recovery and purification of erythromycin.

Type # 6. Aromatic Antibiotics:

The antibiotics with aromatic rings in their structure are regarded as aromatic antibiotics. In a strict since, all the antibiotics containing aromatic nuclei should be considered in this group. However, most authors prefer to treat the three important antibiotics namely chloramphenicol, griseofulvin (Fig. 25.14) and novobiocin in the category of aromatic antibiotics, and the same is done in this book also.

Chloramphenicol:

Chloramphenicol is a broad spectrum antibiotic that can act against Gram-positive and Gram- negative bacteria, besides rickettsia’s, actinomycetes and chlamydia’s. However, administration of chloramphenicol is associated with side effects, the most significant being damage to bone-marrow. As such, chloramphenicol is treated as a reserve antibiotic and selectively used. Chloramphenicol binds to 50S ribosomal subunit and blocks (peptidyltransferase reaction) protein biosynthesis.

Production of chloramphenicol:

Chloramphenicol can be produced by Streptomyces venezuelae and S. omiyanesis. However, chemical synthesis is mostly preferred for the commercial production of chloramphenicol.

Griseofulvin:

Griseofulvin is an antibiotic that acts specifically on fungi with chitinous cell walls. It is used in the treatment of various fungal skin infections. Further, griseofulvin is also employed in the treatment of plant diseases caused by Biotrytis and Alternaria solani. Although the exact mechanism of action of griseofulvin is not known, it is believed that chitin biosynthesis is adversely affected.

Production of griseofulvin:

Commercial production of griseofulvin is carried out by employing Penicillium patulum. The chemical synthesis is less frequently used due to high cost. The fermentation is carried out by an aerobic submerged process with a glucose rich medium. Nitrogen is supplied by sodium nitrate. The optimal conditions for fermentation are—temperature 23-26°C, pH 6.8-7.3, aeration 0.8-1 vvm, and the period is 7-10 days.

Type # 7. Nucleoside Antibiotics:

There are several antibiotics (more than 200 or so) which have nucleoside like structures e.g. puromycin,. blasticidin S. Nucleoside antibiotics have diverse structures and biological activities. Puromycin is used to understand the ribosomal function in protein biosynthesis. Neplanosin possesses antiviral activity. Blasticidin S is a fungicide antibiotic used in plant pathology.

Production of nucleoside antibiotics:

Selected examples of nucleoside antibiotics and the respective organisms from which they are produced are given below.

Puromycin — Streptomyces alboniger

Neplamosin A — Ampullariella regularis

Blasticidin S — S. griseochromogenes

Genetic Manipulations of Steptomyces:

A great majority of antibiotics are produced by Streptomyces sp, a Gram-positive bacteria. Of course, there are some other bacteria (both Gram- positive and Gram-negative) and fungi that can also produce antibiotics. Genetic manipulations in Streptomyces have been extensively carried out to enhance the yield of antibiotics and reduce cost of production, besides developing newer and more effective antibiotics.

Transformation of Streptomyces:

Streptomyces strains exist as aggregates of mycelial filaments and not as individual cells. This is in contrast to E. coli. It is therefore essential that the cell walls of Streptomyces are broken to release protoplasts for transformation (Fig. 25.15). By adding the desired DNA (in plasmids) and polyethylene glycol, the cells can be transformed. These protoplasts are grown on a solid medium to regenerate the cell walls. The transformed cells with desired properties can be isolated for further use.

Cloning of Antibiotic Biosynthesis Genes:

In general, biosynthesis of antibiotics involves several reactions and participation of a large number of enzymes (and of course, several genes). It is rather difficult to clone so many genes. Mutant strains of Streptomyces that totally lack the synthesizing machinery for a particular antibiotic are very useful. Genes from a clone bank can be incorporated into such mutants and screened for the desired properties. This is a lengthy and tedious procedure but has been successfully used for the improved production of certain antibiotics e.g. undeylprodigiosin.

Direct strategies of gene cloning:

It is often possible to identify one or a few important enzymes in the synthesis of antibiotics. From the sequence of amino acids in the enzyme, gene can be constructed, and cloned. For instance, the gene for isopenicillin N synthase from P. chrysogenum has been successfully constructed in this fashion and used for increased production of penicillins and cephalosporin’s.

Genetic Engineering for the Production of Novel Antibiotics:

Genetic manipulations can be done in the antibiotic synthesizing organisms to ultimately produce totally new and novel antibiotics. Wild-type Streptomyces coelicolor genes encode the enzymes to produce the antibiotic actinorhodine. S. violaceoruber produces a related antibiotic namely granaticin. Genetic manipulations can be done between these organisms to produce novel hybrid antibiotics such as medarrhodine A and dihydrogranatirhodine.

The newly synthesized antibiotics are in fact structural variants of the existing antibiotics. As the biosynthetic pathways for antibiotic production and their corresponding genes are better understood, it becomes possible to design newer antibiotics with more efficient action.

Genetic Engineering for Improving Antibiotic Production:

For aerobic microorganisms (e.g. Streptomyces sp), there is often the limitation of oxygen supply that hinders antibiotic production. Some workers have isolated a hemoglobin-like protein produced by the bacterium Vitreoscilla sp. The gene synthesizing this protein was isolated and cloned in a plasmid vector.

The hemoglobin gene of Vitroscilla sp was finally incorporated into Streptomyces. These newly transformed strains have better capacity to take O2 from the medium even at a low concentration. The new strain of S. coelicolor (with hemoglobin gene) was found to produce 10 times more antibiotic actinorhodine than the wild strain, even at a low concentration of oxygen.

Good Antibiotic Manufacturing Practices:

Manufacture of antibiotics is highly commercialized due to a heavy demand worldwide. It is mandatory that detailed clinical trials are carried out before considering the manufacture of any antibiotic. More than half of the antibiotics produced are for human use. If is therefore absolutely essential that each antibiotic produced is safe, consistent and poses no health complications.

Government authorities play a predominant role in regulating the production of antibiotics. These guidelines ensure that correct procedures are followed at each stage of manufacturing the antibiotics. The product produced should be of consistently high quality. Quality of the products should be checked at different stages of manufacture.

It is expected that the antibiotic manufactured should be in the purest form, although 100% purity is not practicable. It is mandatory that the impurities (if any), their quantities, and their ill effects should be made known.


PKSs can be classified into three groups with the following subdivisions:

  • Type I polyketide synthases are large, highly modular proteins.
    • Iterative Type I PKSs reuse domains in a cyclic fashion.
      • NR-PKSs — non-reducing PKSs, the products of which are true polyketides
      • PR-PKSs — partially reducing PKSs
      • FR-PKSs — fully reducing PKSs, the products of which are fatty acid derivatives

      Each type I polyketide-synthase module consists of several domains with defined functions, separated by short spacer regions. The order of modules and domains of a complete polyketide-synthase is as follows (in the order N-terminus to C-terminus):

      • Starting or loading module: AT-ACP-
      • Elongation or extending modules: -KS-AT-[DH-ER-KR]-ACP-
      • Termination or releasing domain: -TE
      • AT: Acyltransferase
      • ACP: Acyl carrier protein with an SH group on the cofactor, a serine-attached 4'-phosphopantetheine
      • KS: Keto-synthase with an SH group on a cysteine side-chain
      • KR: Ketoreductase
      • DH: Dehydratase
      • ER: Enoylreductase
      • MT: Methyltransferase O- or C- (α or β)
      • SH: PLP-dependent cysteine lyase
      • TE: Thioesterase

      The polyketide chain and the starter groups are bound with their carboxy functional group to the SH groups of the ACP and the KS domain through a thioester linkage: R-C(=O)OH + HS-protein <=> R-C(=O)S-protein + H2O.

      The ACP carrier domains are similar to the PCP carrier domains of nonribosomal peptide synthetases, and some proteins combine both types of modules.

      The growing chain is handed over from one thiol group to the next by trans-acylations and is released at the end by hydrolysis or by cyclization (alcoholysis or aminolysis).

      • The starter group, usually acetyl-CoA or its analogues, is loaded onto the ACP domain of the starter module catalyzed by the starter module's AT domain.
      • The polyketide chain is handed over from the ACP domain of the previous module to the KS domain of the current module, catalyzed by the KS domain.
      • The elongation group, usually malonyl-CoA or methylmalonyl-CoA, is loaded onto the current ACP domain catalyzed by the current AT domain.
      • The ACP-bound elongation group reacts in a Claisen condensation with the KS-bound polyketide chain under CO2 evolution, leaving a free KS domain and an ACP-bound elongated polyketide chain. The reaction takes place at the KSn-bound end of the chain, so that the chain moves out one position and the elongation group becomes the new bound group.
      • Optionally, the fragment of the polyketide chain can be altered stepwise by additional domains. The KR (keto-reductase) domain reduces the β-keto group to a β-hydroxy group, the DH (dehydratase) domain splits off H2O, resulting in the α-β-unsaturated alkene, and the ER (enoyl-reductase) domain reduces the α-β-double-bond to a single-bond. It is important to note that these modification domains actually affect the previous addition to the chain (i.e. the group added in the previous module), not the component recruited to the ACP domain of the module containing the modification domain.
      • This cycle is repeated for each elongation module.

      Polyketide synthases are an important source of naturally occurring small molecules used for chemotherapy. [3] For example, many of the commonly used antibiotics, such as tetracycline and macrolides, are produced by polyketide synthases. Other industrially important polyketides are sirolimus (immunosuppressant), erythromycin (antibiotic), lovastatin (anticholesterol drug), and epothilone B (anticancer drug). [4]

      Only about 1% of all known molecules are natural products, yet it has been recognized that almost two thirds of all drugs currently in use are at least in part derived from a natural source. [5] This bias is commonly explained with the argument that natural products have co-evolved in the environment for long time periods and have therefore been pre-selected for active structures. Polyketide synthase products include lipids with antibiotic, antifungal, antitumor, and predator-defense properties however, many of the polyketide synthase pathways that bacteria, fungi and plants commonly use have not yet been characterized. [6] [7] Methods for the detection of novel polyketide synthase pathways in the environment have therefore been developed. Molecular evidence supports the notion that many novel polyketides remain to be discovered from bacterial sources. [8] [9]


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      5.13E: Polyketide Antibiotics - Biology

      a School of Chemistry, University of Bristol, Cantock's Close, Bristol, UK
      E-mail: [email protected], [email protected]

      b School of Biochemistry, University of Bristol, University Walk, Bristol, UK

      c School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK

      Abstract

      With growing understanding of the underlying pathways of polyketide biosynthesis, along with the continual expansion of the synthetic biology toolkit, it is becoming possible to rationally engineer and fine-tune the polyketide biosynthetic machinery for production of new compounds with improved properties such as stability and/or bioactivity. However, engineering the pathway to the thiomarinol antibiotics has proved challenging. Here we report that genes from a marine Pseudoalternomonas sp. producing thiomarinol can be expressed in functional form in the biosynthesis of the clinically important antibiotic mupirocin from the soil bacterium Pseudomonas fluorescens. It is revealed that both pathways employ the same unusual mechanism of tetrahydropyran (THP) ring formation and the enzymes are cross compatible. Furthermore, the efficiency of downstream processing of 10,11-epoxy versus 10,11-alkenic metabolites are comparable. Optimisation of the fermentation conditions in an engineered strain in which production of pseudomonic acid A (with the 10,11-epoxide) is replaced by substantial titres of the more stable pseudomonic acid C (with a 10,11-alkene) pave the way for its development as a more stable antibiotic with wider applications than mupirocin.


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      These authors contributed equally: Bo Pang, Rijing Liao, Zhijun Tang.

      Affiliations

      State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Shanghai, China

      Bo Pang, Zhijun Tang, Shengjie Guo, Zhuhua Wu & Wen Liu

      Shanghai Institute of Precision Medicine, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China



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