Prof. Pinaki Sar
Department of Biotechnology
Indian Institute of Technology-Kharagpur
Lecture - 04
Essence of Metabolic Engineering - Part C
Welcome back to today’s lecture on essence of metabolic engineering part three.
(Refer Slide Time: 00:36)
In today’s lecture, we would be continuing our discussion about the basic concept of metabolic engineering. Today we are going to emphasize on metabolic network and how the metabolic reactions which are present within biochemical pathways are elucidated particularly, what are the steps of enumerating and assessing the pathways within each of the metabolic processes going on in living system.
And those which are involved in converting specific substrate to a target product. In this reference, I would like to emphasize upon the very definition of metabolic engineering wherein we have seen that metabolic engineering has a very unique attribute and that attribute is targeted development of the cell towards a specific product or a specific cellular properties.
So there must be one or more than one metabolic reaction involved towards the production of a specific target molecule or target compound out of the specific substrate, which is provided to the cell. Now during today’s lecture, we are going to
understand how these different reactions which are possibly involved in converting a specific substrate to a target product are enumerated first and then they are assessed.
(Refer Slide Time: 02:32)
And this will be our continuation towards understanding the broad steps and aspects of metabolic engineering which we are doing during these last couple of lectures.
But we have moved forward from the individual enzymatic reaction centric view or understanding towards an understanding of systems level interconnected nature of the biochemical reactions.
(Refer Slide Time: 07:49)
Let us take this example. So here glucose molecule is converted to acetyl-CoA through a part of the central metabolism which is called the glycolytic pathway, the glucose is converted to glucose 6-phosphate and then glucose 6-phosphate to fructose 6-phosphate and then glyceraldehyde 3-phosphate. Glyceraldehyde 3-phosphate is oxidized to phosphoenolpyruvate.
And phosphoenolpyruvate is further metabolized to produce the pyruvic acid. And this pyruvic acid is now subjected to the conversion of acetyl-CoA and acetyl-CoA enters into the TCA cycle. This is the set of reaction or a set of reactions that we see here within these glycolytic reactions. And here we have a set of reactions which are basically the TCA cycle.
Now we should also indicate over here that this particular reaction or set of reactions, which is representing the glycolytic pathway may be constituted by 10 or so reactions and of course, all the reactions are not shown over here in this particular picture, because this is just a diagrammatic representation of the set of reactions.
Prof. Pinaki Sar
Department of Biotechnology
Indian Institute of Technology-Kharagpur
Lecture - 05
Essence of Metabolic Engineering - Part D
Welcome to the last part of our discussion on the introductory topics on metabolic engineering.
(Refer Slide Time: 00:43)
And this is continuation of our earlier discussion where we were trying to build the basic concept of metabolic engineering and particularly the interconnected nature of the metabolic networks that is already discussed. And in this section of this lecture, we are going to learn that the importance of enumeration and assessment of all the pathways for converting a specific substrate to a target product.
And we will be having a brief introduction about metabolic flux and metabolic flux analysis.
(Refer Slide Time: 01:15)
Now enumeration and assessment of all the pathways which are likely to be involved in converting a specific substrate to a target product is one of the major tasks in metabolic engineering. So there are two important aspects. One is the enumeration of the pathways which are likely to be involved and then assessment of these pathways.
So if we want to highlight this particular point, so there could be multiple so let us assume that we have a substrate and the substrate is taken up by the cell and within the cell, so this is the cell boundary if we try to consider it like this. So as the substrate enters inside the cell, so it could be producing the product B, which is actually composed of a number of reactions.
So this is a set of reactions or maybe a particular pathway which is preexisting or we know about this particular pathway. But there could be other several mechanisms by which the same product B is produced. But it might be possible that we are not completely aware about this particular other reactions that which I am now drawing that the alternate reactions like this one, alternate reaction this one, alternate reaction this ones or alternate reaction this ones.
So there could be numbers of such reactions or such pathways, which are potentially capable of converting this particular specific substrate to the targeted product. Now enumeration means that we try to identify all these possible or potential pathways, which are likely to be present in a particular cell or particular cell system and then assess each of these pathway. So there are two points, one is the enumeration.
That is we need to identify that okay, these are the five pathways, so 1, 2, 3, 4 and 5 pathways, which might be converting substrate A or potentially able to convert the substrate A to the product B and then individually we assess each of the pathways. Now this assessment needs certain criteria that based on which we will assess these pathways. So we will briefly talk about that.
So once we identify a specific reaction, so in this case it is the reaction that is the substrate A to product B. Now we will examine all possible reactions or pathways which might be converting A to B and also we will be assessing all those things, all those possible pathways. Now with the goal of overproduction of specific products in mind, when we have in metabolic engineering, we surely will be having a specific goal that we will be producing a particular metabolite or particular product.
Or we will be looking towards improving a particular property of a cell. So when we have a particular property, like particular goal in our mind, like say overproduction of a specific product within using a particular cellular system. The first question that needs to be answered is what pathways can be used to produce the compound of interest. So earlier we were talking about production of ethanol using a simple reaction of pyruvic acid to ethanol.
So if we are interested for example, in producing ethanol in using E. coli or other yeast or other systems, so we need to answer the first question that what are the pathways involved in production of ethanol within a E. coli or yeast system.
(Refer Slide Time: 05:30)
So as I was mentioning, that there could be multiple pathways connecting a pair of specific substrate and target product. Like we have the substrate which is red colored and the product which is green color, and there could be multiple reactions or multiple set of reactions or pathways, which are very clearly evident in case of the glycolysis.
That we were talking about the glycolysis how it is connected to the ED pathway and then pentose phosphate pathway and also with the TCA cycle. So eventually any reaction within these glycolytic pathway is under the influence and under control of multiple reaction. So we need to actually elucidate or enumerate all these multiple pathways which could be responsible for producing ethanol or maybe pyruvic acid if we take for an example.
So the first point is that there could be multiple pathways involved in conversion of a particular substrate to a particular product. And we need to enumerate or identify each of these pathways. Now often the interconnectedness of the metabolic reactions like in the glycolytic reaction we have seen the interconnectedness of metabolic reactions in such that the number of potential pathways linking substrate to the products is actually huge.
So if we want to elucidate all we will be surprised to see that actually from pyruvic acid to ethanol, this could be a very unique reaction, but the flux or the flow of carbon up to pyruvic acid, it is controlled by many reactions within the glycolytic pathway
that ultimately leads to the production of the ethanol. So interconnectedness of the metabolic reactions is also to be included over here.
And there could be actually as we mentioned, there could be huge number of potential pathway. So actually, when we looking we will started looking into the whole genome based data apart from biochemical assessment of different pathways that how a particular yeast or particular bacteria could might be converting substrate like glucose or any pentose sugar or other hexose sugars into ethanol, some number of pathways were elucidated.
But as soon as we started looking into the whole genome sequence of the organism, we were surprised to see there are actually more number of potential pathways which are actually potential pathways that they could actually link the substrate or to the desired product. Now these pathways, first is the all the pathways are need to be identified.
Some of the pathways will be absorbed chemically that by biochemical assay, by detecting the specific enzymes we can confirm that these pathways are actually indeed present in a particular species or particular cellular system and they are working towards a substrate to product formation.
And some of the pathways are actually potential pathways that under certain circumstances, these enzymes will be expressed or the genes encoding these enzymes will be expressed and these pathways might be working towards the production of the desired product.
(Refer Slide Time: 08:45)
Now these pathways are first to be identified that is called enumeration of these pathways. Okay, these are the pathways, but they are to be assessed against each other. And other criteria means there are multiple criteria that we are going to discuss briefly.
That when we have a number of pathways, which are connecting a specific substrate to a specific product, all these pathways need to be assessed in terms of the thermodynamic feasibility of each of these pathways under biological conditions. Particularly when we integrate the genome annotation data with the mathematical modeling and then try to build the metabolic network and look into the interconnections.
Often we are able to discover or identify a number of novel pathways or potentially new pathways, okay, under which might be working under different environmental conditions or under different structures. Now all these pathways, maybe 5, 6, 10 or maybe 50 pathways are to be assessed in terms of thermodynamic feasibility or whether they are thermodynamically favorable or not that within the cellular system the biological conditions are very precise conditions.
So within the biological conditions whether these all these pathways that we are able to find out are going to work or not. The second point is that the intermediate metabolites, we can actually find out a pathway which is converting our substrate to our desired product. But the intermediates might be toxic as we have noticed and we
will discuss in some of the other classes that intermediates are often found to be toxic to the cell.
So we could see that, for example, well known substrate for amino acid production with chorismate. So this chorismate could actually lead to the production of phenylalanine to tyrosine and ultimately to tryptophan. So numerous other than these three particular conversion, numerous novel biochemical pathways involved in conversion of the substrate to these products were elucidated.
And the thermodynamic analysis of this pathway suggested that native pathways are thermodynamically more favorable. So as I was mentioned before, that it is not only enumerating or proposing new pathways that we have observed that this pathway could be present in a particular system for converting a substrate to a product, but the thermodynamic favorability or thermodynamic feasibility of such reactions are to be established.
So using this kind of computationally supported database, they were able to show that it is many of the pathways which are proposed, these pathways are not thermodynamically favorable, while the native pathways which are existing or preexisting and known for conversion of these chorismate to tryptophan tyrosine phenylalanine are actually thermodynamically more favorable.
The pathways generated involved compounds that exist in biological and chemical databases. So when they looked into the entire set of data projected or predicted by this computational process, they found many of the intermediates, which are predicted by the system are existing in biological and chemical databases.
That means they are biologically produced and chemically they are relevant, they are already there in the database, suggesting novel biochemical roots for these compounds and the existence of biochemical compounds that remain to be discovered.
So based on their observation, this group, when they studied this, they found that novel pathways and novel reactions to discover such kind of intermediates, which the intermediates are already there, but the pathways are not known, could be discovered or could be identified.
(Refer Slide Time: 17:57)
So in number of similar approaches using such kind of systems were used subsequently for evaluating the pathways for 3-hydroxy propionate and 1,4-
butanediol. So all are industrially very important metabolic engineering relevant products.
(Refer Slide Time: 18:09)
Now we are going to talk briefly about metabolic flux. Metabolic flux, we are going to talk in terms of defining it and metabolic flux analysis. But before entering into these things, we just want to reiterate that metabolic pathways which we are possibly talking for some time now and we have gained some level of understanding because it encompasses a number of reactions and sharing kind of common metabolites within it.
So metabolic pathways and fluxes are at the core of metabolic engineering. So we need to identify and understand the metabolic pathways and also characterize the metabolic fluxes within the studied system and that is identified to be a core of metabolic engineering. So after metabolic network or interconnected systems level understanding, this metabolic flux analysis is another very important aspect.
(Refer Slide Time: 19:12)
Now here we need to very clearly define metabolic pathway, what is it actually? Metabolic pathway we often use the term in a very general sense that it is producing some kind of product out of a substrate and which is basically catalyzed by a number of enzymes etc. Now it is defined as any sequence of feasible and observable biochemical reaction steps connecting a specified set of input and output metabolites.
So it clearly says that it is basically a sequence of reactions. So it is a pathway. So it is a pathway that means like a glycolytic pathway or tricarboxylic acid cycle, which we called as the TCA cycle, it is a pathway. So that is actually a sequence of reactions. So a number of reactions are there and product of the first reaction is likely to be the substrate for the second reaction and so on and so forth.
And finally, the product is produced. Now this these reactions which are actually representing the pathway must be feasible and observable biochemical reactions. And they must be interconnecting a specific set of input and output.
(Refer Slide Time: 20:31)
So for example, we are coming back to this picture where we were talking about the glycolytic reaction and along with glycolytic reaction, we are able to see that how acetyl-CoA can be converted to or pyruvate can be converted to ethanol.
And also we see that how amino acid sorry the how the TCA cycle is connected to these acetyl-CoA and even phosphoenolpyruvate can be connected to the TCA cycle by producing oxaloacetate. And the entire set of this TCA cycle is again connected to amino acid biosynthesis. So it represents a number of
But eventually, these all reactions need to be validated before proposing a new pathway about that particular reaction.
(Refer Slide Time: 25:59)
Now the pathway flux. So once the pathway is well defined, the pathway flux can be defined. So pathway flux or metabolic flux of a particular pathway, because it has to be specific for a pathway or specific for a reaction is defined as the rate at which the input metabolites are processed to form the output metabolites.
And if we consider this particular reaction, where you have A to B, is A is the substrate and B is the product in this reaction. And in that case, if we consider flux is J, then the flux J is equal to the rates of individual reactions at steady state.
So whenever the reaction is steady state we will find that the intermediate metabolites are adjusted to concentration that make all the reaction rate equal and therefore J is equal to the rates of the individual reactions, which is V1 = V2 = V3 = V4. Now there may be transient states when the actually transient conditions when the individual reactions are not at steady states. So we have to understand that also.
So the reactions must be reaching into a steady state that is a fundamental consideration before calculating the metabolic flux.
(Refer Slide Time: 27:17)
Now metabolic flux analysis is this method, which is enabling us to understand the flux of the entire metabolic pathway. So the primary method for analyzing this network is the platform which is called metabolic flux analysis. So through the metabolic flux analysis or MFA, we are able to analyze the flux through the entire network of the metabolic pathway.
The metabolic fluxes are determined under different conditions. So in MFA, we generally allow the cells to grow under control condition as well as under different altered condition like with varying oxygen concentration, varying substrate like glucose to other hexose sugar or maybe pentose sugar with more change in pH or change in oxygen concentration as I mentioned to.
And then try to find out how the flux of carbon for example, if you are studying the carbon metabolism is changed or altered, when we change the conditions. And by creating which are called actually perturbations by such perturbation studies, we are able to find out what are the points where actually the flux is being controlled or regulated.
(Refer Slide Time: 31:29)
So metabolic flux analysis includes the determination of intracellular fluxes. So after the substrate is taken up, so it is started with historically with 13 C carbon for example. So once the labelled carbon is taken up inside the cell, we are able to measure different metabolites where this label 13 C carbon is there.
So basically we determine the intracellular flux that what is the rate of flow of movement of these labelled substrates for example, along with these analysis of factors affecting the flux distribution. Factors could be indigenous factors like other substrate, other metabolites, cofactors reducing equivalent like NADH, NADPH, FADH2 or give energy sources like ATP or GTP etc.
Or they could be factors which are collected from the external environment or the growth medium. And then collectively, all these data will be represented in terms of the metabolic map that we will be able to figure out that how this metabolic flux is being changed as the reaction proceeds from the starting reaction one to the last reaction, reaction L.
So basically the MFA or metabolic flux analysis combines data on uptake and secretion rates. So how the substrate, initial substrate is taken up and how the products are released or transported out. Biosynthetic requirements, what are the associated requirements other than the substrates. Metabolic stoichiometry how the reactions are progressing and the stoichiometric characteristics of these reactions.
And quasi-steady-state mass balance on metabolic intermediate to determine the intracellular metabolic fluxes.
(Refer Slide Time: 33:21)
So final outcome of this metabolic flux calculation is a metabolic flux map like as presented over here. And you can see the 13 C flux map of aerobically grown E. coli cell. So similarly, in the same study the Chen et al has also shown how it is changed when the same E. coli cell is grown under anaerobic condition.
As you can see here, that this thickness of the arrow indicates the relative proportion of the, relative proportion of the flux which is actually carried out by this particular stretch of the reaction. So this is the entire glycolytic reaction up to pyruvic acid. So you can see that it is, the flux is relatively higher when glyceraldehyde 3-phosphate is converted to phosphoenolpyruvate and phosphoenolpyruvate to pyruvic acid.
And then finally, under aerobic condition, a strong flux is there that pyruvic acid is going towards acetyl-CoA. Because, why this strong flux is there during aerobic condition, because if we move to anaerobic condition, we will see there is a decline over here. Because this acetyl-CoA is going to enter into TCA cycle. So that is the kind of flux but in E. coli it is very interesting system.
We will discuss about E. coli in detail in the other lectures. That this huge amount of flux is also there towards acetate formation from acetyl-CoA. So pyruvic acid to acetyl-CoA. Acetyl-CoA supposed to feed this entire TCA cycle. But at the same time
acetyl-CoA is also responsible largely to produce acetate. So in case of E. coli that is true.
So you can so such kind of flux and we can see the relative importance of each of the reactions and also can identify that suppose we want to improve the flux towards TCA cycle, okay. We can see that relatively less flux is moving towards TCA cycle. So what could be the reason? Because up to this huge amount of flux is coming. Then suddenly the flux is getting reduced over here.
So what could be the limiting factor or what could be the governing factor, controlling factor. Who is responsible for splitting the flux into acetate side or this TCA cycle side. So these are some kind of discussions and some kind of understanding that we actually gain once we identify a particular target.
(Refer Slide Time: 35:42)
Now information about these metabolic flux can be enormously helpful in identifying the critical branch points in the pathway. Like in the previous slide, we could identify a branch point where the flux coming from pyruvic acid to acetyl-CoA is distributed towards TCA cycle and acetate. In case of E. coli, a large fraction of the flux is going towards acetate rather than in towards the TCA cycle site.
So we can identify the critical branch points towards in this pathway. Discover unusual pathways in less characterized species. So instead of E. coli suppose, we are
working on some novel bacterial strain or newly isolated organism, we can identify what are the unusual pathways if we study these kind of flux analysis or flux maps.
Define the maximum theoretical yield for the synthesis of products from complex integrated pathway producing and consuming multiple cofactors and intermediates. So what could be the maximum yield out of the given moles of substrate that is provided to the organism that can be calculated based on the flux analysis. And ultimately, this flux analysis defines the flexibility and rigidity of the enzymes and branch point of the networks.
So when we talk about these kinds of metabolic reactions, we see there is a flux going down and there are flux branch points where the flux is being splitted between different part of the pathways. And we are finally able to define or identify the nodes or the branch points, which are flexible or rigid.
And based on the this identification of their flexibility and rigidity of enzymes and branch points, subsequent and further strategies were developed and ultimately those strategies are implemented through genetic engineering tools.
(Refer Slide Time: 37:37)
So during this particular part of the lecture, we have used the following references.
(Refer Slide Time: 37:46)
And to summarize this part of the lecture. So basically we have highlighted the first question to answer for achieving the goal of overproduction of a specific product, what pathways can we use to produce a compound of interest, because there could be multiple pathways connecting a particular substrate to a particular product.
Now when we have such kind of multiple pathways, often predicted by different metabolic network analysis, we need to understand this all these pathways must be enumerated and assessed against multiple criteria, including their thermodynamic feasibility, favourability, the toxicity status of the intermediates and expression of the genes and enzymes.
We have also defined or tried to understand the metabolic pathway and what is the usefulness of the feasibility and observability of the individual reactions. And finally, again a brief idea about what is metabolic flux and what is the importance of metabolic flux analysis. Thank you.
But the point also is to be highlighted that some of these metabolites are intermediate products like hexose 6-phosphate like fructose 6-phosphate or fructose 1,6 bisphosphate or even glucose phosphate, glucose 6-phosphate. These are also involved in other pathways like the hexose 6-phosphates could be the substrate for the ED pathway, which is very one of the very important pathways in microbial system or the other very important pathway which is the pentose phosphate pathway.
So these glycolytic reaction which is supposed to be discussed or emphasized only with respect to conversion of glucose to acetyl-CoA or towards the pyruvic acid, which ultimately connecting it towards the with the TCA cycle.
So how these enzymes are involved in connecting particular substrate to a particular product and then a particular product might be a substrate of another enzyme. So the entire map of the reactions, how the reactions are interconnected, the pathway chemistry and the stoichiometry that is the, it is connected towards the theoretical yield of the products, everything need to be elucidated very clearly.
(Refer Slide Time: 27:50)
Now here if we present a very simple representation of this metabolic network development, where you can see a set of related metabolic reactions can be represented. So these are the individual reactions like M1 is converted to M2. Substrate M1 is converted to product M2. M2 is the substrate for another enzyme E2 converting it to M3. And thereby M3 to M4 and M2 can be also converted to M4.
So there are four enzymes which are catalyzing four metabolic reactions in a given system. So this is just an example of how the metabolic networks are actually developed or built in a very simple manner. So we can see that these reactions or these individual reactions can be connected like M1 to M2 and M2 to M3. And M2 can also be converting to M4. And M3 can also be converting to M4.
So this is a simple or simplistic view of the network with respect to four particular metabolites or four particular products and intermediates and substance representing a metabolite. Now this type of networks can be represented in multiple ways. So we are just going to present here two very general and very simple ways of representing the metabolic work. One is the metabolite centric metabolic work.
Now with this level of description, which is also parallely coupled or supported by mathematical modeling etc., so we are able to analyze the complete metabolic system. So like this in this case, the simple schematic diagram represent the same reaction like A to D. Now here you can see that how actually A is converted to D and what are the requirement of cofactors, what are the requirement or the potential reactions where the different byproducts are produced.
And how the different substrates are actually processed and how they are actually interconnected.
(Refer Slide Time: 37:10)
Okay, so during this particular part of this lecture, we have covered the following literature and we of course, followed the textbook which is the metabolic engineering textbook, but some additional reference papers are also used.
(Refer Slide Time: 37:24)
And in summary, in this lecture, we have discussed the individual enzyme reaction to systems of interconnected biochemical reaction and thereby an enhanced perspective of metabolism and cellular function is obtained from such systems of interconnected reaction. And finally, the metabolic networks which are basically understood in a very fundamental or preliminary levels and their developments that how from reaction to the networks are progressed.
And some of the requirements before producing these or before coming into this metabolic networks are also presented. Thank you.
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