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Module 1: Biosynthesis and Cellular Transport

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Macromolecules and Growth Metabolism

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Metabolic Engineering
Prof. Pinaki Sar
Department of Biotechnology
Indian Institute of Technology-Kharagpur
Lecture - 06
Review of Cellular Metabolism
In today’s class on metabolic engineering, we are going to talk about cellular metabolism and we will discuss the broad aspects of cellular metabolism and review it with respect to metabolic engineering applications.
(Refer Slide Time: 00:52)
So during this lecture and in continuation with that, we will be highlighting the basic aspects of cellular metabolisms and other components and important parameters and processes within it. Overall objective of this lectures would be to synthesize a biochemical concept in the framework of cellular metabolism.
(Refer Slide Time: 01:21)
Now water is the most abundant cellular component across all the different type of cellular systems. Nearly 70% of the cellular component is represented by water and rest that is 30% or nearly 30% is constituted by different cellular materials which actually represents the dry cell weight biomass. Now this 30% chemical constituents, they are distributed mainly among different macromolecules.
(Refer Slide Time: 02:08)
And these macromolecules are mostly represented by a large number of molecules including the protein, different type of RNAs, including the ribosomal RNA, transfer RNA and messenger RNA. DNA, which includes both the chromosomal DNA as well as the plasmid DNA. Lipids, lipopolysaccharides, peptidoglycan, glycogen and different other soluble pools.
Now the diversity and abundance of these molecules, all these macromolecules are pretty abundant. Like if we look into the number of protein molecules, different kind of protein molecules present in an E. coli cell that could go more than, up to more than 1000 different kinds and the total number of protein molecules could be as high as 2.36 billion types.
The type of ribosomal RNA compared to other things are relatively less because as we know there are only three principal types or main types of ribosomal RNA so far found in cellular systems, which is only three. Transfer RNAs are quite high up to 60. And messenger RNAs are also significantly high because it is representing the transcripts or the genes which are being expressed at any point of time particularly during the active growth phase of the cells.
Other molecules including the lipids, lipopolysaccharide, etc., generally present in varying concentrations.
(Refer Slide Time: 03:55)
Now synthesis and organization of these macromolecules into a functioning cell occur by several independent reactions. These biosynthetic reactions which are independent to each other include the nucleotide biosynthesis, lipid biosynthesis, protein biosynthesis or the peptidoglycan biosynthesis.
And there may be many other biosynthetic reactions which are leading towards different other macromolecule synthesis as required by the cells. All cells are not
having equal requirement. So apart from the common requirements of different nucleotides, lipid and protein peptidoglycan etc., there might be a number of specialized molecules required by different other cell types.
Now interestingly although all these biosynthetic reactions or the reactions which are leading towards the formation and the assembly and the production of these polymer or these macromolecules these are quite independent. But interestingly, these all biosynthetic reactions are connected to a particular pool of precursor molecules which are small metabolites molecules, which are continuously providing the resources, the chemical and other resources for the biosynthesis of large number of macromolecules.
So the point of interest over here is that there are large number of independent reactions, which are producing different type of macromolecules like the nucleotide, lipid, protein etc. However, all these biosynthetic reactions are originating or utilizing fewer number of small metabolites or small chemical compounds, which are called precursors.
(Refer Slide Time: 05:58)
Now the precursor for the synthesis of these macromolecules are small as I mentioned before, rapidly used pool of low molecular weight compounds. Now these precursors are constantly replenished by biochemical synthesis from metabolites ultimately derived from glucose or other carbon sources. Now let us discuss this particular point.
For example, if the cell is utilizing glucose as its carbon source or carbon and energy source that glucose is taken inside the cell that is the glucose transport process. So following the transport of the glucose, a series of reactions, a series of biochemical reactions are taking place and these biochemical reactions are converting this glucose or similar type of complex organic molecules to a set of small organic precursor molecule.
So these are small molecules like two to three carbon molecules or sometimes even four carbon molecules, but generally it is three to four carbon molecules. These are small molecules which are considered or called as precursor molecule, because they are used as the starting molecule or molecule of major importance in initiating the biosynthesis of all other macromolecules.
For example, a large number of reactions which we were discussing in the previous slide, which are basically the independent biosynthetic reactions, these biosynthetic reactions eventually lead to the production of the synthesis of a large number of macromolecules and their organization. So a large number of synthesis and organization related organic processes or biochemical reactions, which are leading towards the synthesis of macromolecules are basically utilizing the small set of precursor molecules.
(Refer Slide Time: 08:08)
Now there is a fundamental biological reason that why it is often necessary to make a large number of genetic modifications to alter cell metabolism, particularly with
respect to metabolic engineering, when we are trying to improve a native cell, this is the native cell condition, okay. This cell we are trying to improve with respect to production of a particular molecule, maybe the earlier the production was very low.
So one of the first step towards the anabolic reaction is the conversion of the organism carbon sources to precursor metabolites and from that precursor metabolite or number of precursor metabolites synthesis of the monomers and other building blocks are achieved. The next is the synthesis of the macromolecules from the monomers because once the monomers like the amino acids or the nucleotides are produced, then the synthesis of the macromolecules by polymerization reactions are achieved.
And finally, once the polymers are produced, then the assembly of the macromolecules are required because the macromolecules which are produced are to be converted or to be structurally organized to form the required or functionally or structurally active molecule. So assembly reactions actually enable the cells to produce the required cellular structure require the molecules in proper structural configuration.
(Refer Slide Time: 33:34)
So if we look at the same metabolic map where we were observing the central carbon metabolism, so this is the part of the central carbon, entire stretch of the central carbon metabolism coupled to the TCA cycle and also coupled to the pentose phosphate pathway. If we briefly look into this, we will be able to get a kind of a brief understanding about how these.
So this stretch of reaction you can assume that partly it is the catabolic reactions, because the energy is continuously generated. So it is oxidized and the energy is consumed and energy is generated over several reactions. And also you have the number of metabolites produced. Now each of these metabolites could be utilized by the anabolic reactions, where you have the complex macromolecules are to be synthesized.
(Refer Slide Time: 34:26)
Now these catabolic reactions are actually linked tightly with the anabolic reactions, because on the one hand the catabolic reactions like if we write the catabolic reactions which is basically the organic molecules are processed to produce the smaller molecules. But during this process, we are producing the ATP and also we are producing the reducing equivalent which is basically the NADH+H+.
Now the anabolic reactions, the anabolic reactions, they are utilizing these smaller molecules, many of the smaller molecules, which are going to be used as the precursors. So they are converted into precursor molecule and these precursor molecule they are utilizing these reducing power because they need the electrons and also they need the energy from this ATP and ultimately, the complex macromolecules are produced.
So from smaller precursor molecules we can produce the complex macromolecules through the anabolic reactions, which require the supply of the small molecules, supply of the energy and supply of the reducing power of NADH H+ which are provided by the catabolic reaction. So essentially catabolic reactions are intrinsically connected to the anabolic reactions.
(Refer Slide Time: 36:05)
Now overall structure of the cell synthesis because we are more concerned about the synthesis of the macromolecules because that is often one of the major interest in metabolic engineering point of view.
So we have seen these catabolic, set of catabolic reactions where a number of biochemical reactions including the glycolysis, pentose phosphate pathway, the TCA cycle is enabling the production of these major metabolites which are the substrate for the anabolic reaction and also the energy resources like the ATP and the reducing equivalents.
And these set of reactions, the whole bunch of catabolic reactions are actually connected to the anabolic reactions, because the precursor metabolites are synthesized out of the many metabolites into metabolic intermediates, which are being produced over here. And with the input of the energy and with the input of the reducing power, which are produced from the catabolic reactions, the building blocks are produced and then building blocks are assembled into the different macromolecules.
(Refer Slide Time: 37:10)
For today’s class, we are we have basically used the metabolic engineering our textbook and a few review papers by the review paper of Nielsen and Keasling published in Cell that is engineering cellular metabolism and partly we are also going to use the Prescott’s microbiology book by Willey et al. Thank you.
Metabolic Engineering
Prof. Pinaki Sar
Department of Biotechnology
Indian Institute of Technology-Kharagpur
Lecture - 07
Review of Cellular Metabolism - Part B
In today’s metabolic engineering class, we are going to discuss about the metabolic frameworks operating within the cellular system.
(Refer Slide Time: 00:43)
pressure condition, then it would be require more mechanisms to or implementation of mechanisms to maintain that. That is called the homeostasis.
So when such situations are there, so cell might be requiring even more energy to spend to maintain and to achieve a kind of homeostasis. Now the amount of energy and reducing power needed for growth can be calculated once we know two very important things. So the amount of energy and reducing power which is subjected to the growth condition or the environmental conditions in which the cells are exposed.
But they can be determined by determining the composition of the cell under specified growth condition. The specified growth condition is a very important term over here. So if we change the growth condition like from the medium to pH to temperature to the oxygen diffusion, any kind of environmental change might lead to a change in composition of the cell materials and hence, the energy requirement of the cells.
Now details of the pathway of biosynthesis and polymerization, that information is also required. So in fact during the process of all the metabolic pathway assessment and including the flux, MFA flux analysis and all those things, we analyze this biosynthetic reactions, biosynthetic process, particularly how the carbon flux is processing within the metabolic reactions.
(Refer Slide Time: 37:03)
Now how is growth fuelled in microorganisms particularly? This is very well-known fact but we want to just quickly revise this in case of heterotrophic organisms which are relying on complex organic compound as their source of carbon. So there could be chemoheterotrophs and or could be photoheterotrophs. The chemoheterotrophs the source of organic compounds while the source of energy is also organic compound.
So in case of chemoheterotrophs like for example E. coli or yeast, it is basically the carbon substrate which is we provide like yeast extract or maybe the lactate or acetate or the glucose molecules, these organic molecules provide the source of carbon and also the source of energy. However, for the photoheterotrophs the carbon source is the organic compounds like the glucose or other carbon compounds, organic compounds, but the source of energy is the light energy.
So they harvest the light energy and they utilize the carbon from the organic carbon. In case of autotrophs there are also two types of autotrophs, the chemoautotrophs and photoautotrophs. The chemoautotrophs they use source of carbon. In case of autotrophy is obviously carbon dioxide because they rely on fixing their own carbon. So in both the chemo and photoautotrophs it is the carbon dioxide which are which is there as a carbon source.
However, the source of energy is different in case of chemoautotroph. The source of energy is the inorganic compounds like the ammonia or the water or even iron or sulfur compounds, which are highly reduced compound if we oxidize or the cell oxidize them and get the electrons out of them and those electrons are used in the cellular metabolism. In case of photoautotrophs, it is the light energy.
So light energy is utilized to actually derive the electrons from water or H2S or other molecules.
(Refer Slide Time: 39:09)
Now two interesting facts are there with respect to the growth metabolism which is connected to this framework of metabolism. Metabolism is diversified to process specific nutrients available in the environment, yet all lead to conserved internal order and chemistry.
What I was referring to earlier that over the evolutionary period microorganisms, mostly microorganisms and then other eukaryotic organisms too, they try to maintain this framework of metabolism starting from the nutrient sources like the carbon energy sources to the fueling reactions up to the assembly reactions. Now the cellular system can begin with any of the starting materials.
Like in case of heterotrophs, no matter whether in the medium you have glucose or succinic acid or maltose or yeast extract, the end products will be always the same, the same set of molecules which the cell actually requires. Obviously, the kind of secondary metabolites or other metabolites which the cell might not be using for its own purposes.
Sometimes in metabolic engineering, we try to exploit that properties of the cells that is different. But otherwise, for the regular compounds, the framework is very well designed and it is like a kind of a funneling reaction. Like all different kinds of substrates can be accommodated and the cellular system will be able to run smoothly using those different set of substrates.
(Refer Slide Time: 40:46)
Although there are certain organisms which are called picky type of organisms or they are very selective about their nutrients, but apart from that, generally the heterotrophic microorganisms are very versatile with respect to their nutrients and nutritional sources. The second very interesting point about this framework is that the cells couple chemical reactions to drive the essential process.
So there are, I mentioned there is a large number of chemical reactions involved in this framework okay, which actually culminate in growth and reproduction or maintenance of the healthy cell type. So cell phenotype or cell physiology will be maintained in a healthy state if the cell is not at least not divided. Now production of living cells depend not only on the chemistry of the metabolic reactions, like the kind of reactions.
There are large number of reactions going on inside the cell, but also on the cooperative interplay of these reactions, this is another very interesting thing. So while the first interesting thing about the framework was the acceptability of different substrates. You can start, the cells can start, particularly the heterotrophic organism, they can use any of the different kind of organic carbons.
And then still achieve the similar set of complex well-structured compounds, which are required for the cellular function, but at the same time, a strong cooperative interplay of numerous reactions going on the cells. Because here we are not representing the reactions, which are just showing the broad flow of processes.
So these arrows are not to be considered as reactions, these are only the flow of the framework that the fueling reactions is now feeding the biosynthetic reactions. But here we can have few 100 reactions operating on that. Now how these reactions are regulated, coordinated as per the requirement of the final products like the cell is trying to divide or cell is not trying to divide, cell is planning to maintain the non-growth condition.
So feedback loops and other control devices operating in very orderly manner, okay. We will be talking about that in another lecture. And it has been found that hundreds of reactions are regulated to ensure that they function cooperatively in a grand synthesis. The grand synthesis is the synthesis of the required molecules, and only the required molecules which are essential for the cellular function and cellular growth.
(Refer Slide Time: 43:14)
Now the next one that we are going to talk today is that biochemical pathways are connected, because we have already talked about the metabolites and we have talked about ATP and reducing power. So biochemical pathways within a metabolic setup can be connected or interconnected by participation of intermediates.
Many intermediates like hexose phosphate or pyruvic acid or glyceraldehyde 3-phosphate for example or oxaloacetic acid or ribulose 5-phosphate or different other such small molecules, they help the cell to engage or connect multiple pathways with each other. And there might be a question like how these are actually selected that
which metabolites will be involved in more reactions and which metabolites would not be involved in more reactions.
So this is that is not the purview of today’s lecture. But the second important connecting compound is the cofactors including the ATP and similar high energy currencies, NADH, NADPH and FADH2. Now continuous formation and utilization of these cofactors like continuously the ATP molecules are produced and they are also consumed, because in some of the other slides, we have seen that some reactions are thermodynamically not favorable.
So they might be requiring the input of ATP to make them favorable, make them proceeding towards the desirable site. Some reactions might be reducing reactions or requiring the electrons from NADH or FADH2 or NADPH.
Now on the one hand, these are continuously being produced by the fueling reactions and the other side these are continuously being consumed by the different biosynthetic reactions or assembly reactions or even some of the activation type of reactions which are there in the catabolic set of reactions itself.
(Refer Slide Time: 45:11)
Now the organization of the biochemical pathways. So there are we are mentioning that there are many pathways inside the cellular system like a tiny E. coli might have more than a thousand or so, close to thousand reactions going on and there are a large
number of pathways. Pathways mean we have metabolic pathways, we have defined a set of well-organized sequence of reactions by feasible and observable events.
So these pathways are generally organized because in metabolic engineering, we need to organize them in order to understand and in order to modify them appropriately. So these reactions, biochemical pathways are organized based on their chemical properties, they are based on their physical properties and also based on their characteristic times.
So chemically through sequential conversion of metabolites as we generally see in case of a biochemical reaction series, that a particular substrate is converted to a particular product, which is an intermediate and then this product is converted to another product, why because these two reactions are interconnected and so like glycolytic reactions or pentose phosphate pathway.
These are called chemically organized reactions like pentose phosphate pathway itself is a chemically well-organized reaction and Embden-Meyerhof-Parnas pathway is another very well organized set of reactions. So there could be chemically the reactions are organized. Physical organization of the chemical reactions or biochemical pathways can be understood best when we look at the fact that the different set of reactions are operating at different parts of structure within the cells.
Particularly in case of eukaryotic organisms like yeast we can understand that for example, the TCA cycle operates in mitochondria, beta oxidation of very long chain fatty acids are happening in the peroxisomes. Or DNA and RNA synthesis are happening inside the nucleus. So set of biochemical pathways are very well organized with respect to their physical location, some reactions are happening in particular location within the cell itself.
(Refer Slide Time: 47:34)
The third and most important category of organization is organizing the reactions based on their characteristic types. Now before we define the characteristic times we need to understand that different reactions although there are numerous reactions as we have been talking within the cells, there are numerous reactions operating. Now different reactions are operating at different timescale.
Some reactions are very fast like an allosteric control is very fast or nutritional changes, which are very slow. So there are reactions with all kinds of variable timescale. Some reactions are fast, some reactions are slow some reactions are very fast and so on. So we are going to discuss briefly about that and then we will define the characteristic times.
Now when considering on the reaction pathway, we should focus or select the reactions with comparable timescales only. So when I when we are looking at an entire stretch of reaction system or a cellular metabolism, we should make selection about selecting the reactions or the particular reactions based on their characteristic time and we should consider the reactions with comparable timescale only.
Now the faster reactions which are like in this case the allosteric reaction is much faster than the mutational reactions. Now faster reaction can be assumed to be at equilibrium upstream of a slow reaction or give rise to steady state for the metabolites downstream of a slow reaction step. And much slower reactions.
Much slower reactions, reactions like mutational change if we consider with respect to enzyme control can be ignored as they operate on a completely different timescale. Like when we are expecting that the due to the change in reactive configuration or the change in the environmental condition there might be change in enzyme activities, the control mechanisms or the kind of enzyme synthesis etc., might be altered or changed.
Now during that particular consideration, we should actually ignore changes like mutational changes for example, which are truly very slow and may not be included in this analysis.
(Refer Slide Time: 50:01)
Now the relaxation time, which is actually the present the characteristic time of the reaction. The characteristic time of the reaction approximated as a first order process is called relaxation time. For relaxation time of a given reaction is basically the characteristic time of the reaction approximated as the first order process. Now reaction relevance within the given time frame is assessed by comparing the relaxation time of the various reactions.
(Refer Slide Time: 50:28)
For example, here we see that schematic comparison of relaxation time of different processes operating in a living cell. Now with respect to the x axis is the relaxation times and as you can see changes like the mutation, the enzyme induction, they are particularly the mutational changes are pretty slow taking 10 to the power 6, 10 to the power 9, 10 to the power 12 seconds, very slow process.
Compared to cell growth like cell growth we can have 10 to the power 3 to 10 to the power 5 or so. That is the timeframe within which the cell growth will be completed, few hours for example. Allosteric controls or the enzyme controls are relatively faster depending upon the type of the controls and type of the enzyme mechanisms it can go up to less than a microsecond to a second only.
mRNA control, how the mRNA production transcription is controlled, that is a matter of only an hour or so within that or less than an hour within that the mRNA are produced or controlled. In comparison to the cell growth or mRNA control, mass action or the diffusion of the molecules inside the cells are pretty fast. Like they are less than microseconds or so.
(Refer Slide Time: 51:47)
Now when we are comparing different reactions, there could be some reactions which are on absolutely different timescales and they are referred as in this case, frozen reaction. Reactions with much larger, much larger in the sense 10 times or more relaxation time than that of the system of interest. Like if we are considering cell growth with respect to that mutational changes are surely the frozen reaction.
Because they are operating at a much larger 10 times or more timescale. So with respect to cell growth, mutational changes can be considered as frozen reactions and they may not be included, they may not be included within the study. On the other hand, there could be pseudo-equilibrium reactions, because these reactions are having relaxation time much smaller than that of the system.
We were referring to this earlier. So compared to cell growth, the allosteric controls are actually pseudo-equilibrium reaction because they are having significantly lower relaxation time compared to the cell growth.
(Refer Slide Time: 52:52)
Finally, the conclusion remark out of this characteristic time segment is that metabolic processes can be simplified. We know that there are a large number of metabolic reactions and metabolic processes are happening inside the cell. So that can be simplified significantly by ignoring reactions and pathways operating on timescale outside the time range of interest.
If we are, for time being if we are working on the enzyme regulation, we may ignore even this, even the partly the cell growth also. If we are considering the mRNA expression, however, cell growth will be very relevant because the timescales are overlapping. But the mass transfer or the diffusional factors might be on a kind of a pseudo-equilibrium. So we may not consider them, may not include them.
So when we have large number of reactions to investigate and then understand that how they are working together so we may use the scheme like we can use the chemical organization or the physical organization or the relaxation time based concept to simplify the overall metabolic process.
(Refer Slide Time: 54:04)
So in today’s class, we have highlighted the framework of metabolism emphasizing on the different components and how these important parts of the framework are interconnected and interdependent, and the usefulness of this growth metabolism or the energy supply during the non-growth metabolism as well. Organization of the biochemical reactions and the concept of relaxation time is also covered. Thank you.