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Module 1: Metabolic Network Analysis

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Metabolic Network and Systems Biology

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Metabolic Engineering
Prof. Amit Ghosh
School of Energy Science and Engineering
Indian Institute of Technology – Kharagpur
Lecture – 12
Introduction to Metabolic Networks
Welcome to metabolic engineering course, so today we are going to learn a very interesting topic that is the introduction to metabolic network.
(Refer Slide Time: 00:45)
So, in this lecture I will give some background of metabolic networks like bioenergetics and then measure metabolic pathways and then laws of mass action and the regulatory of metabolic network and then followed by 1 dimension and 2 dimension and notation of genome sequences. So, this will give you enough background to actually constructing the metabolic network it will give an introduction. So that you can understand or you can make the metabolic network on your own.
(Refer Slide Time: 01:18)
So, first I will start with and that the living cells require energy, so all of you agree that living cells require energy for biosynthesis transport of nutrients motility and maintenance, then how the cell gets energy? The cell actually gets energy from the catabolism of carbon compounds mainly the carbohydrate that we consume the microbial cell they consume only glucose.
So, they consume this substrate which is a carbohydrate and then and through catabolism on the left hand side you can see that the catabolism. So, through catabolism they are actually well to decompose the sugar into small molecules and in the process they get energy, the ATP, NADPH or genetic these are the energy molecule and this is required for all the processes the cell perform.
For example, the biosynthesis of protein, DNA, RNA, transport of nutrient, motility maintenance also all these functions are actually the cells are well to perform because of this energy which is generated from catabolism. So, catabolism is basically the intercellular process for degrading a compound into smaller molecule and also produce energy for the cell. It will not only produce a smaller molecule but also produce that is the essential part of the cell to produce energy.
So that cell conform all its functions and then also it produces key metabolites. So, on this region you can see that these are the key metabolites produced through catabolism and that is super phosphate PEP, pyruvate, acetyl coenzyme, alpha ketoglutarate, succinic, coenzyme,
oxaloacetate and so on. So, there are eleven key metabolites present inside the cell, these are the precursor for many of the compounds.
Or these are present for almost in every cell right from microbial cell to the mammalian cell. And this catabolism the metabolic pathways are almost similar for all kinds of organisms right from bottom to top. And these molecules remain as a precursor molecule for anabolism. So, anabolism is a process which actually uses this small molecule and produce complex molecule.
For example, amino acids, nucleotide, fatty acid and those are required for the growth of the cell not only that, we have protein, RNA, DNA, these all macromolecules are actually synthesize through a process known as anabolism. This is already known I am just summarizing. The anomalism is involved in the synthesis are more complex compound that is from glucose to glucose and that is the storage of energy and these processes require energy.
So, the first I told catabolism which produces energy and the anabolism which required energy. So, whatever energy you get in this process, gets utilized in anabolism. So, this way the cell is actually creating its own energy and also utilizing that energy for many other processes. For example, formation of protein, DNA, RNA these are macromolecules synthesis these are the macro molecules which are present inside the cell and because of that we see the cell is growing the cell biomass is increasing.
So, we have I do not know whether you have seen how the cell is growing in a flask. So, if you have done culture or not but generally the cell grows like this. We have a curve like this and then it goes initially with time, this is x axis we have the time and the y axis we have growth and growth is measured in terms of OD optical density would the culture inside a spectrophotometer and then you can measure the optical density to know that how much it is concentrated.
So, initially when you start the culture here you will find the OD is almost point you start with 0.1 OD and then cell grows and the exponential phase we get increased OD which is around more than one 1.52 and then slowly it saturates. After some time you see that there it has reached the stationary phase. So, this is the exponential phase of the cell and this is the stationary phase.
So, this is you already know yes I am repeating it again, so that you make your revise this thing again. So, the cell grows and you see the growth or the biomass is increasing, OD is increasing when that means the concentration of the media is increasing at which the cell is growing. And this is because the cell is making protein, RNA, DNA membrane organelles. So, these are these cells are making through and these give rise to the growth. And then you see the cell biomass is increasing and you can measure the cell biomass using OD.
(Refer Slide Time: 06:41)
So, next you come to the bioenergetics part like how the protein DNA require large amount of ATP. For making protein DNA cell requires around 39 millimole ATP per gram of protein then RNA we need 7.4 millmole ATP per gram of RNA and then for DNA we need 11 millimole ATP per gram of DNA. So, these are the ratio of ATP and the energy requirement we have in the cell.
So, the cell you know the protein is the important part of the cell which perform most of the function and that require the synthesis of protein require a lot of ATP molecule. The production of biomass enzyme for biofuels syntheses suppose you want to make a compound inside the cells through metabolic engineering that also requires energy. So, everywhere whatever you are going to make for example I am telling the biofuel production for the plasmid.
Suppose, you are putting a plasmid inside the cell or any synthetic scaffold you are putting inside the cell which is not natural you are expressing heterologously. Then also it not only
consume carbon building blocks the carbon is needed but also it require energy 2 things are required. First the carbon is required the carbon in the sense that glucose it consumed that converted into carbon.
And then those carbons goes into the building block of this molecule, suppose you are producing biomass or when you are producing enzymes inside the cell. You put the plasmid inside the cell and then that get amplified inside the cell and it from enzyme or protein or any synthetic scaffold you are putting. So, it not only requires carbon but also request energy molecule.
So, this you should keep in your mind that these are the 2 things you need because when I say that metabolic engineering or manufacturing any product inside the cell will require not only carbon but also energy. Second large amount of ATP needed to be consumed to support or cell maintenance. As you know apart from building biomass enzymes biosynthesis, you also need ATP to support the cell maintenance.
Processes including energy spilling microbial motility cell component repair re-synthesis macro molecule all these processes require ATP. And third the synthesis of biofuel molecule needs ATP and NADPH, for example fatty acid require 7 ATP. So, supposes you want to make fatty acid inside the cell 80 / 7 ATP and 14 NADPH to convert acetyl coenzyme A into 1 fatty acid molecule that is permitted that is C16 carbon as number of carbons present in permitted is 16.
So, this enter process not only require carbon but also ATP molecule or NADPH molecule those are energy molecules which are required by the cell for making this compound in the form of a fatty acid inside the cell. So, these are the constraints the cell have first of all the carbon you are putting in and also the energy this way. If the cell is not having sufficient energy that is ATP molecule then yield will go low.
So that you should know even there you have sufficient carbon in the media but if the cell is actually not have energy then the yield of production will go low. So, these are the 2 factors you should look at when you try to make any compound.
(Refer Slide Time: 10:24)
So, these are the major metabolic pathway, you must have gone through already those pathway first is the glycolysis process which is basically glucose is consumed and then 2 molecule of pyruvate it is made and you get 2 ATP molecule. So, in this process you get only 2 ATP molecules through glycolysis and then pyruvate goes into TCA cycle. So, the conversion of glucose to pyruvate is also known as a to EMP pathway.
And these empty after pyruvate is produced for EMP pathway it is transferred to the Krebs cycle or TCA cycle were in balance of energy plus the pyruvate is converted into acetyl coenzyme and you have 1 molecules of NADH. Since we have 2 molecules of pyruvate therefore 2 molecules of pyruvate will give 2 molecules of NADH. And then we have the overall TCA cycle reaction which is shown over here the acetyl coenzyme A, again it is converted into 3 molecules of NADH.
So, each molecule of acetyl coenzyme A is converted into 3 molecule of NADH and then also produces CO 2. So, this I am showing just to tell you that how many energy molecules are produced per molecules of glucose.
(Refer Slide Time: 11:52)
So, if you can estimate that the total theoretical yield that is 38 molecules are produced from one molecule of glucose. So, among these 38 molecule glycolysis and TCA only produced 4 molecules of ATP and remaining 34 ATP molecules are generated through oxidative phosphorylation. So oxidative phosphorylation is the process where you get a remaining 34 ATP molecule and what is oxidative phosphorylation? This is the process of forming ATP from the electron transport chain is known as oxidative phosphorylation.
And the term P / O that is the ratio is used to indicate the number of phosphate bond form for each oxygen atom used as an electron acceptor. So, we generally assume the P / O ratio as 3 when I do when the ratio is 3 then 3 ATP phosphates is made from each proton transport by the electron transport chain. So, it is using this we can assume that for NADH the P / O ratio is 3 that is, for every NADH molecule you generate 3 ATP molecules.
And for FADH you generate 2 NDAH to 2 ATP molecule, so this is the ratio you can use if the P / O ratio goes down then you get less or ATP molecule. So, the here eventually when the P / O ratio is 3 then the number of ATP molecule for every NADPH is 3 and FDAH 2 2 ATP. So, the combining this many number of ATP molecule you will see that 38 ATP molecules are produced for every one molecule of glucose.
Now under anaerobic condition the energy metabolism is insufficient and cells often secrete acetate to overcome the ATP shortage. If the biosynthesis requires a large amount of ATP oxidative phosphorylation become a key source for satisfying the ATP demand. So, the oxidative phosphorylation will become a very important mechanism by which the ATP
maintenance or the ATP requirement is satisfied when you want to synthesize a biofuel or a chemical or any by product from the cell.
So, it is always keep the P / O ratio to 3 but if it goes down then you there is a deficiency in energy, so you are really goes down. These are the constraints you have are the metabolic burden you have inside the cell when you try to make new molecule. You know anyone who tries to make a new molecule there is a metabolic burden inside the cell.
(Refer Slide Time: 14:45)
So, then, in addition to the high ATP demand imposed by the biofuel synthesis pathway or biofuel synthesis pathway inside the cell metabolic flux analysis studies have revealed that the overexpression biosynthesis pathway significantly reduces ATP maintenance expenditure and the metabolic burden in engineered micro further causes poor respiration efficiency. So, as you had new metabolic pathway inside the cell or any heterologous pathway you are adding inside the cell then the ATP demand increases.
So, significantly activity when increases and then because of that we have a poor respiration efficiency that is a phosphorylation process P / O ratio goes down from 3 it become 1.3. So, that also becomes a constraint in this the hosts suffer from severe ATP limitation the effort to increase carbon availability to biofuel synthesis will be useless. So, if there is cell is undergoing severe ATP limitation.
Then what will happen and as much as you keep substrate for the cell that is useless because the cell is actually suffering through ATP limitation. Many metabolic engineering approaches
were applied to improve carbon efficiency are effective regarding the carbon to biofuel in low result in low productivity strain. So, you try to give as much carbon to the media but he actually said is not able to consume because of the energy limitation.
Because they lacks ATP inside the cell and that is why your biofuel productivity or the production level give rise to low productivity of the strains. So, the yield goes down so try to this will be given, so that the carbon yield and the energy efficiency have to be carefully balanced, we have to balance these 2 things the carbon yield and also the energy efficiency that is the amount of ATP production said the cells should balance each other. So that you have better yield in terms of carbon, these 2 things you have to keep in mind when you design new cell form for production of new compounds through metabolic engineering.
(Refer Slide Time: 17:03)
So, this is this slide which is taken from Professor Jay Keasling metabolic engineering class notebook and then you can see the first equation is the glucose is consumed inside the cell and it is producing 686 kilo calorie per mole. So, this much energy is produced when glucose is consumed. And then we have the ATP how many ATP molecules are produced you can see from glycolysis it is producing 2 ATP molecule.
So, this way the coordinated action of the various gene product giving rise to the expression of some gene and this is the integrated function, this genetic circuit is very crucial to understand the cell physiology or to understand the systems perspective of the cell you need to understand the gene genetic circuit present inside the cell. The function of genetic circuits is diverse that is DNA replication, translation glucose to pyruvate formation all requires different kinds of genetic circuit.
The information processing or this or any kind of cellular processes require the understanding of the genetic study. So, here from the genomics from the sequence that you identify how the genetic circuits are actually connected to genetic circuit remain very crucial in systems biology. And then that you can get directly from the DNA sequence, from the DNA sequence you identify how many genes are there and the gene products are there and then how many genetic services are present?
(Refer Slide Time: 27:34)
So, the application of genetic circuits you can see in energy production and then information processing all the cells at process which I already told you energy, this is the metabolism where the currency metabolites are formed and how genetic circuits are present which are involved in metabolism, how the metabolite form because of different genetic study that you need to identify manually, it is helping in metabolic engineering.
Because metabolic engineering deal with removal or adding on new genes but, if you keep on adding and new genes or new things inside the cell, what will happen that your genetic circuit is getting disturb because the genuine circuit is unique for a given cell. So, understanding the genetic circuit will help a better in metabolic engineering, it can also help genetic circuits are used for transcription and translation and post translation modification.
Also even it is used for cell division and cell adhesion, cell differentiation that gives rise to tissue engineering. So, all these dynamics inside the cells are actually given by the genetic circuit. The genetic circuit that you get from the; sequence the genome sequence and try to identify how many genetic circuit the cell may have.
(Refer Slide Time: 28:59)
So, now, I asked a question if every molecule inside the cell is replaced over time, is it still the same cell or not? Suppose, you are actually replacing 1 cell at a time; removing replacing 1 cell at a time. Now, the question is that is it the same cell or it will be a different cell. Similarly, if every cell in every molecule inside the cell is replaced, first I told that this diagram is a cell that will have many cells and you are replacing 1 cell at a time with another organism.
And you want to say that whether the organisms remain the same or not. And in the other case, you are actually having the each of the molecule inside the cell you are replacing. So, each of the molecule which are basically metabolite replacing with and try to say that whether the cell remain the same or not because you are changing the cell component and also in the organism you are replacing the cell. Now, the question is that whether it will remain the same organism or it will become a different organism and what is the answer for you? Let me let us guess what could be the answer for this?
(Refer Slide Time: 30:12)
And the answer is that yes. So, we the interconnection of the biological network, if every molecule is replaced over time is it remain the still same cell or it will become a different cell or if you replace the cell from the human organism will it remain the same cell or it will be a different organism, so, it will remain a different segment we will remain the same cell even if we change the component of the cell like metabolite it will remain the same even if you replace the cell of the organism.
It will remain the same organism because the interconnection of the biological component that is the blueprint or the circuit diagrams of the cells are taking the center stage in biology. So, if you replace a component, you are not changing the interconnection. So that is the circuit diagram that is a genetic circuit present inside the cell it remains the same that is the blueprint of the biology and that is genetic circuits are more important that is what I want to say.
That the genetic circuits are more important and that gave rise to the emergence property or the cell that is the emergence of systems biology. If you know the genetic circuit that actually gives the wiring diagram of the life, so even if you change the component or the cell, this is not going to change the organism. So, then and then what is the nature of link between the component in a biological network, what kind of link do you have in a biological network.
The molecule in molecular biology the chemical reactions are basically the link between the metabolites and then in tissue we have the gap junction and then in sociology, we have friend whether to personally how the 2 persons are connected to each other that is through friendship or they are married. So, this way, you have a network when you say the network any network has node and edges.
The connection between 2 nodes that is a connection between 2 persons in sociology is connected through that kind of relationship, you have. Similar in in the biological network, you have the biochemical reaction network, the nodes are basically the metabolites and the link between the nodes are the reaction. So, this way, the networks are present in every field and you have to understand how they are connected. The connection between the; metabolites are important, rather than the metabolite itself that is what the circuit diagram means.
(Refer Slide Time: 32:47)
Some key feature of biological network, what are the functional state and the properties of the biological network, they are constrained by physiochemical laws selected by evolution, they have many states and they have a sense of purpose that is, they have an objective function. The objective function is basically the survival. So, every microbial cell have a sense of purpose, they want to grow more and they and they want to survive.
And these are the way you can define the objective function of the microbial cell. And the biological networks have in many states, many equivalent states and they are selected by evolution and they are also obeying laws of physics and chemistry as well. And the functional state and the properties of the biological network are defined by the different functional state present inside the cell.
(Refer Slide Time: 33:39)
So, here I as I told the biological networks are have many functional state, what does it mean? So, to and to give an analogy, I took an example. This is an example to understand how t many equivalent state I told that biological networks have many equivalent states and that actually help the biological cell to be more robust, they are more they can survive in many condition because they have many equivalent state.
If you will that is why the microbial cells are very robust compared to any other multicellular cell. And the biology of microbial cells are easy to grow because they have many, in overall like biological, they do not have many state and that can be understood by using these example where you can see suppose you want to print this document. And then by for printing the document, you have many options and either you go to the print option and print it, or you can use a command or you can actually have a button here on the tab.
So, 3 ways you can print these documents. So, you have multiple state equivalent options and these equivalent options are present inside the cell also, the cell can choose many ways to perform the same function. It can choose many ways in this cell, it can form many network circuit diagram that is generic circuit to perform the same function. So, this makes the biological network much more robust and they can survive in many conditions because the networks are many equivalent states are present.
(Refer Slide Time: 35:16)
So, in conclusion, we learned about what is systems biology? The systems biology basically study a biological component and how they function as a whole. So and then why there is systems biology, the systems biology actually deal with biological component and high throughput technology because of huge amount of biological data through high throughput technology which make system biology feasible.
And the potential applications of systems biology are there in industry, medicine, agriculture and etcetera. As a huge amount of application, you can see through systems biology, how can you implement this system analysis, what do you need to know to system biology deal with systems analyzes and for that you require the component that is the nodes and how they are interacting and that there is a connection between this node so that it can form a network.
What are the key feature of a biological network which as I discuss, it has an objective function that every network has an objective function and the multiple solution state exists and this is achieved through evolution. So, every biological network, the microbial cell, or you can consider any biological network, they evolve over time. So that the best interconnectedness of the world wiring diagram are taken up by the cell through evolution.
So, there you man that gave rise to maximum survival, the cell can survive more by considering that particular network. So, this lecture, we I gave you some introduction of about the systems biology which will help you to understand more about the biological network.
(Refer Slide Time: 36:58)
So, these are the books that you can follow the systems biology the properties are reconstructed network written by Bernard Palsson and also you can refer to some of the paper from Bernard Palsson that is the challenges in Silico Biology. 2 dimensional annotation of genome. So, in the next lecture, we will learn more about the metabolic network, how we are going to use this system biology to construct the metabolic network. Thank you. Thank you for listening. Hope you understand systems biology in this lecture and will see you in the next lecture.