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Essence of Metabolic Engineering

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Introduction to Metabolic EngineeringGood morning. Today we will be starting the first lecture of metabolic engineering course. Thank you for registration. Thank you for taking this course. This course will be taught by me myself and Professor Pinaki Sar from biotechnology department. We will start with the introductory lecture, basic introduction about metabolic engineering, how the course will go. Overall we will give you an idea what will be covered in this course.(Refer Slide Time: 01:03)And we will take this introductory course into following points like course outline, what we will be learning from this course in several weeks and then followed by the principles of metabolic engineering, what are the steps followed in metabolic engineering and then followed by importance of metabolic engineering.Why people do metabolic engineering, what is the need for metabolic engineering that will be discussed in a broad perspective. And then followed by challenges of metabolic engineering. So what are the problem, what are the issues when you do metabolic engineering and then finally, the application of metabolic engineering we will be learning in this lecture.(Refer Slide Time: 01:48 )So the textbook we will be following, the name of the book is metabolic engineering, principles and methodologies. And this book is actually written by Gregory Stephanopoulos from MIT USA coauthored by Aristidou and Jen Nielsen. So this was published in 1998 and is considered as one of the best book for metabolic engineering.(Refer Slide Time: 02:18)So in this course, myself and Professor Pinaki Sar from Department of biotechnology IIT Kharagpur will be teaching this course and then the and we will cover mostly following topics. It will start with introductory to metabolic engineering, basic concepts, scopes, and then application, overview. For example, cellular transport processes, fueling reaction, and that will be covered in first week.Followed by second week we will have some introduction to MATLAB and then cellular metabolism overview, the biosynthetic reaction polymerization, growth energetics, regulation or metabolic pathway. Further on this third week we will talk about reconstruction or genome scale metabolic network. Genome this also this is this will have many sub topics as well.Followed by we will have the metabolic flux analysis and metabolomics where we will learn about flux balance analyzes, flux variability analysis, and then flux map and also we will learn about metabolomics. Then on the fifth week, we will learn about experimental determination of fluxes. That is through isotope level substrate, isotope mapping metrics, isotope distribution vector, those are the subtopic you will be learning.And then on week six, we will be learning about application of metabolic flux analysis. On seven week, seventh week we will learn about experimental tools used for engineering metabolic pathway, and then how to control gene expression, genome editing tool, adaptive laboratory evolution and reverse engineering. And on the last eight week, we will learn about some example of manipulation or metabolic engineering bioenergy, bioremediation healthcare and agriculture.So we will cover as much new topics toward the end where metabolic engineering is applied. So we will start with some basic introduction, learn about metabolic pathways and then we will go to advanced topics like genome scale model, metabolic flux analyzes and then experimental determination of flux analysis like that in CMFA. Then application of metabolic flux analysis so and also advanced genome editing tool which is used in synthetic biology.You will also get an idea how you can actually apply this in metabolic engineering.(Refer Slide Time: 04:55)So come to the basic like what is metabolic engineering? So many of you are actually learning for the first time have no idea what is metabolic engineering. So I have defined in such a way as metabolic engineering is a process for modification of specific biochemical reaction of the organism so as to produce required amount of the desired metabolite through recombinant DNA technology.Considering advantage over the other chemical synthesis route, this area of biotechnology is likely to revolutionize in future. So the main idea about the metabolic engineering came that you have to produce some metabolite or a compound or it can be biofuel, it can be some chemical industrially important chemical which you want to make in the industry. So it has a huge potential in the sense and most of the chemicals you get from raw petroleum.Any byproduct you want to make in that you have to choose the organism itself. Now these organisms are chosen based on the availability or the knowledge available for that organism. So generally this can be yeast or E. coli.Because yeast and E. coli are very well known in terms of their molecular biology tool, in terms of their genetics, we know most of the things not all of them, but approximately around 50 to 60% of the organism is known, because it has been studied for several decade. For each time talking about model organism like Saccharomyces cerevisiae.And yeast is also metabolically engineered to produce different chemical used for bio production. And then E. coli is also used. So mostly this host organism can be these two organisms, Saccharomyces cerevisiae and E. coli. And then you have to select the pathway. The pathway is selected based on the molecule you are targeting. Suppose you want to make succinic acid.Suppose you want to make some molecule which is produced in the organism but not in a great amount. So the metabolic engineering deal with production of the chemical in huge amount not just, if naturally it is producing it may not be sufficient for industrial production. So for that you have to increase the production. Either you have to increase the production or you have to make a chemical which is not produced by the organism.That also you can do. So both way you have to, if you want to improve the production, which naturally the organism is producing or in other way you can. So this is the yeast. You choose this host organism and then you choose for the metabolic pathway. Suppose, I look for some amino acid, so then I have to improve the amino acid like the glutamate. So glutamate you want to improve the production.So we choose that pathway and then you build some castle. So these are the genetic parts you want to put in in the host organism. So this is the native organism. So you have the native organism here and then and this is the, you are building the parts. Soin the building parts is also part of synthetic biology. So and the metabolic engineering also deal with synthetic biology.Many of you heard about synthetic biology. Synthetic biology deal with different biological part or the genetic parts. For example, promoter, gene, coding sequence, terminator. So there are many things in the you can include in the part assembly. So the part assembly is a process where you design the pathway. And then you integrate or you put it in the cell. So before it is actually put in the pathway you actually design it in the computer.So this way if you design or buy those gene and put it into the organism through genetic engineering tool like CRISPR Cas9. So here the concept of genetic engineering is coming, where the recombinant (Refer Slide Time: 26:23)Historically, the synthetic biology synthetic component that is designed build of metabolic engineering appeared first. So in the metabolic engineering the syntheticcomponent actually came first where you can apply molecular biology tool. The main enabling technology is the actual genetic engineering technology that through which you are actually adding new genes.So in this diagram, you can see that this is suppose you consider this as a E. coli cell, E. coli. Why you have chosen E. coli? Because E. coli is easy to handle. You know most of the things in E. coli, most of the when I say that most of the thing you know that means you know most of the genes in E. coli. Not all of them.E. coli is the most well studied organism, one of the most well studied organism where you know most of the molecular biology tool and you can actually add new genes from different sources like from plant you can get a gene. So you can insert the gene in the inside the cell from the plant. Also you can from human being, from also from other microbe you can insert and all the other mammalian organism also you can have those gene.So through genomics, because of the revolution in genomics also you are able to do this, because lot of sequences are available right now. If you go to NCBI database, you will get several 100 organisms are available, whose metabolic pathway you want to put it in E. coli because that particular product you want to make inside the cell. For example, I give one example like you have heard about anti-malarial drug, right?Artemisinin is actually anti-malarial drug which is used to treat malaria patient. And also we saw a Nobel Prize for this molecule, the artemisinin drug where the person got the Nobel Prize for extracting this compound from this plant. So the plant is used to actually extract the drug. But this plant is actually not very easily available.So that is why one scientist what he has done, he actually inserted the gene which is required for making the artemisinin drug and put it into a microorganism and tried to make that compound through genetic engineering. So through genetic engineering, he inserted those metabolic pathway for the production of the drug.So this way, you can actually design the cell using the synthetic biology or the genetic engineering tool can be applied, where you can include two or more different sources.You can even include gene from two different sources. So two or more different sources you try to insert the gene in the microorganism like E. coli and try to generate the compound.For example, the artemisinin drug was produced from yeast, Saccharomyces cerevisiae not E. coli. So this is just to tell you that you can get genes from various sources and during the design you try to insert those metabolic pathway in the chromosome of the cell so that you have a production.(Refer Slide Time: 30:09)And then the analytical component or metabolic engineering is basically deal with measurement. So here I am showing one component that is GC mass, the gas chromatography set up for mass spectrometry. So it has a combination of gas chromatography and mass spectrometer, where you try to measure the compound, how much the cell is producing. This is a very important technique we use in metabolic engineering, where you try to measure the amount of product the cell is producing.And this is a check, primary check which you can actually know whether your metabolic pathway is working or not. So learning this is also part of a metabolomics, where you try to actually measure the metabolite, not all the metabolite inside the cell, but some of the metabolite you can measureusing GC mass.(Refer Slide Time: 31:05)nstitute the metabolome of the cell.The metabolic network are defined by pathways. The metabolic network is basically this each node is basically a metabolite. And the connection between the two metabolite is basically a reaction. This is the understanding this network is actually any kind of network has two component, one is node and another is edges. Edges are basically the reaction.So the and the metabolic networks you can identify this network directly from the genome sequence, because of the well-known technique which are available which you will learn in this course that is basically part of systems biology. So in this course, we will learn a little bit about systems biology, where you will be able to actually identify this kind of metabolic network or metabolic pathway directly from the sequence.And the flux and also once you have the metabolic network, you can actually calculate the flux. Flux is the rate of turnover of molecule through a pathway. Suppose this reaction, there is a reaction going from here and now we want to know how much carbon is flowing in this reaction. So in this reaction, that is also you can calculate and identify how much carbon it is going through.And the flux is regulated by the enzyme in a pathway. And this flux is also regulated and that is why you need to learn about the regulation of the metabolic pathway. So in this course, we will also learn about little bit about the regulation of the metabolic pathway, how much carbon it is flowing and how much it is regulated, that also you need some idea for doing the metabolic engineering.Not just the pathway, you also need to know how these metabolic pathways are regulated inside the cell. This way, you would be able to actually have an idea how the carbons are flowing inside the cell, because this is very well connected. So this also we will learn about in detail in subsequent classes.(Refer Slide Time: 35:42)So the metabolic engineering is like managing a traffic. This slide gives you an overview of what is metabolic engineering. So in the left hand side, you can see that we have a metabolite and then there is a reaction, this metabolite is converted into this compound that is from glucose to D fructose. And then the and each metabolites are connected through a reaction and there is an enzyme.So this is the enzyme for this reaction, this is the enzyme for this reaction like that, they are connected in a network. It is even compared to traffic. Nowadays, if you see the Google map if you open a Google map you will see that which roads are actually congested. So the red regions are actually are congested. That is there is a traffic jam. So we do not go in that pathway.And because of the Google you get an online distribution of the traffic jam on the road. And this may happen similar this thing you can see in the metabolic network, where the carbon atoms are the people. So you can consider the traffic and compare it to the metabolic network and the metabolites are the location. And enzymes are the road and the railway they travel on.So enzymes are basically the road. So if you do not have enzyme then what happen this metabolite may not be converted into another metabolite. And the metabolites are the location. So suppose you want to go from here to here, these metabolites are basically the location and the roads are basically the enzyme. This way, you can actually understand what is metabolic engineering.The carbons and atoms are the people. So the one people is going through this road and then from going from one place to the other, that is from metabolite 1 to metabolite 2, and then it is going through an enzyme. So this is what the analogy which is used in metabolic engineering to understand it better.(Refer Slide Time: 37:57)Now I take a small example of metabolic engineering of succinic acid. So as you know the succinic acid is industrially a living compound, where is very useful solvent in industry, the succinic acid. It is used in many purpose. It has an industry is a high commercial value, and there are many ways you can make succinic acid. So using metabolic engineering by engineering the cell also you can make succinic acid.Here I have shown the metabolic pathway of succinic acid. So the sugar is entering the cell. It is coming from outside going inside the cell and then it is converted into pyruvate. And then from pyruvate to acetyl coenzyme A. And then from acetyl coenzyme A to it goes to TCA cycle. And from TCA cycle you are getting the succinic acid. So this is the product you are looking for.And these are the intermediate metabolite weight. It has first converted into pyruvate and then it is should be converted into acetyl coenzyme A and then it should enter the TCA cycle and then it produce the succinic acid. Now my question is that if I want to improve the production of so I want to make improvement in succinic acid production.So what I should do to improve the production of succinic acid? So the straightforward way you actually you improve suppose you want to improve the production of enzyme 1. As I told in the previous slide that the enzyme production should improve. So to improve the enzyme production E 1, do you think that the succinic acid will improve?Suppose I increase the production of enzyme inside the cell by up regulating that gene, which is responsible for enzyme 1. So do we get the succinic acid production increase because now enzymes are produced more this way? The question is that if I increase the enzyme 1 should I get the improvement of succinic acid production also as well directly proportional or not.So whatever your answer the answer is no, simply the answer is no. Because if you improve the production increase the production of enzyme 1, the succinic acid production may not go high because from pyruvate the amount of acetyl coenzyme A produced is from pyruvate it is going to acetyl coenzyme A. And this acetyl coenzyme A is going into many other path.We can see that acetyl coenzyme A is going into fatty acid pathway. Then it is going to monoterpene. And then only small portion of the flux goes into TCA cycle. So the inside the cell even though you are producing more acetyl coenzyme A but only the fraction of acetyl coenzyme A which is produced more is going into TCA cycle. Andthen inside the TCA cycle small fraction of the TCA cycle flux is going into succinic acid.So this is a systems biology problem, because the metabolite which is produced inside the cell may not be converted totally into succinic acid that is the carbon. Suppose the carbon from the sugar is entering the cell then the and then the moment the enzyme 1 is more if you upregulate the enzyme 1 then more acetyl coenzyme A will be formed.But the more acetyl coenzyme A may not enter into the TCA cycle. It may go into another reaction that is the idea. That is why metabolic engineering when you do this is a system biology problem. You have to understand with a system perspective where you have to consider the metabolic network and you have to see how much flux is actually going into different pathway.So when you make when you are interested to make some product like succinic acid using biological system then these are the hurdle you will be facing, it is not very straightforward. And since you are improving succinic acid you are not happy with the wild type strain. So the wild type may not produce enough succinic acid. So that is why you are doing the metabolic engineering.Using bioengineering, you want to upregulate this enzyme 1. So that is why you need a modeling technique or how actually the carbons are flowing and going into succinic acid. So this problem can be addressed using a systems biology perspective, where you want to understand the how the carbons are flowing into different network pathways and you have to be very clear.(Refer Slide Time: 42:46)So another these are the challenges in metabolic engineering that you have to understand the problem in systems biology perspective. Not only that, if you know the metabolic pathway, and then you want to actually make that pathway inside the cell for that you need genetic parts. So these are the genetic parts which are available, the coding sequence, terminator, then plasmid, prima.So all those thing you have to you should know. And this is very much easier when you consider E. coli or Saccharomyces cerevisiae. But if you go to non-conventional organism, then the biological parts are less in unknown organism. So this the parts you have to know when you do the metabolic engineering. And then not only the parts, you should know the molecular biology tool of that organism.So whether that molecular biology tools are available because these genetic parts you have to actually insert in the organism. For that you need the molecular biology tool like transformation, cloning you would be able to do for that organism. And also you should know the genetic circuit. The genetic circuit is basically the regulation.How those metabolic genes in which you are inserting inside the cell are how they are regulated, that you should know. These are the challenges in metabolic engineering in terms of biological parts, and also how these metabolic parts are actually regulated inside the cell once you insert inside the cell. And also their molecular biology tool you should know.So this if you know all those things, then your metabolic engineering is easy. Otherwise, it can be very time consuming if you do not know many of this things. The first is the metabolic pathway. You should be knowing the metabolic pathway and also the biological part and also the regulation of the metabolic genes which you are inserting or how it will affect when you insert a new gene.These if you know this three thing, then it is easier. Otherwise you have to do lot of study to understand this thing before starting the metabolic engineering.(Refer Slide Time: 44:59)So now I come to the need for metabolic engineering. The rapid increase of global population and the living standard combined with limited availability of conventional chemical industry to reduce the production cost followed by detrimental effect of environment made metabolic engineering driven biotechnological manufacturing technology the only alternative and the choice of the future.Because right now, as I told before also that we mostly depend on petroleum derived products. So most of the chemical product you use is actually derived from petroleum resource. And metabolic engineering can help in this regard, because metabolic when you do metabolic engineering of the microbes, it is basically renewable way which is environmental friendly.This is one of the factor for which the government is spending lot of money, a lot of industries are also spending money to actually make this product available throughmetabolic engineering. And other thing is basically, the cost of production. So the conventional way what industry is producing these chemical maybe may not be cost effective and also not environmental friendly.So to overcome these challenges, you need metabolic engineering driven biotechnological manufacturing technology that can be the choice of the future and is alternative procedure, which you can get it higher production through metabolic engineering. To overcome this challenge metabolic engineering provides the biotech industry with tool of rational strain design and optimization.This brings about significant shift in manufacturing costs, and yield of desired product which is suitable for the environment. So using the metabolic engineering tool you can actually design the strain. Strain is basically nothing but the microbe, which you are actually engineering. You have to optimize that metabolic pathway so that the cost of production decreases.So a lot of engineering, several rounds of engineering you have to do through DBTL cycle that is design, build, test, learn cycle to reduce the cost of production. So one is the cost and another is environmental friendly. These are the two main motivation for metabolic engineering so that you get more yield, more product you can produce in this way through the so that you do not have to depend on the conventional chemical industry.Why? The conventional chemical industries are actually not environmentally friendly. So you have to save your environment. When you do metabolic engineering, you are actually making this product in a environmental friendly way. And also you can improve the cost of production. So put together these are the challenges or these are the benefits you have for metabolic engineering.(Refer Slide Time: 47:42)And the application of metabolic engineering. Also you can see that you can make lot of things, like you can make solvent, food ingredient, fuel, polymers, lubricant, pharmaceutical, pharma company, flavors, fragrance. So right now using metabolic engineering, this metabolic engineering is applied in this field, where you can actually produce lot of things, not just fuel, solvent.Several things can be produced using metabolic engineering. And the main, metabolic engineering is also used for fuel production for renewable sources, convert biomass into chemical. It can be used for therapeutic compound. There is a lot of funding from US Department of Energy and a huge investment came from companies like British Petroleum, Chevron, and then Bill Gates they are investing lot of money for this kind of product.Because this is really an alternative to the petrochemical industry. From petrochemical industry also you get most of the compound, but it is not environmental friendly. That is a reason that we are getting lot of funding from other organizations who are supporting this kind of production. Also you can think of many other companies like Amyris, LS9, Lygos, Genometica. They are actually dealing with metabolic engineering.(Refer Slide Time: 49:12)So some of the industries that some of the success the industry came from by applying metabolic engineering. For example, the genomatica. So genomatica has produced bio-butane or butanediol. Biobutanediol can be used for many purposes, where it can be a chemical feedstock that replaces petroleum based product. It is a precursor molecule for many compounds, which converts sugars into BDO that is butanediol through microorganism.And it right now it is producing 30,000 tons of renewable BDO per year. And it is actually benefits for local agriculture economy. A lot of people are also employed. And this is, the genomatica company is based on California and the company which is actually producing in Italy and the plant has been opened in 2016.(Refer Slide Time: 50:13)Many other product for example, artemisinin, which I have already told, it is a drug we use for malaria treatment is also produced from Saccharomyces cerevisiae, where it took almost 10 year. Right now the company which is actually involved in production of artemisinin is the Amyris company. And this product took almost 10 years which goes from laboratory scale to the industrial scale.And the project cost around $50 million. And then we have the PDO is actually a fiber used for many purpose, which you can make from, it took almost 15 years to actually make this compound available in the market and the cost of the project is around 130 million and right now the product is manufactured from DuPont. DuPont is actually making this compound.(Refer Slide Time: 51:14)In conclusion, the metabolic engineering is actually a four step process; that is design, build, test and learn. So metabolic engineering as you know is a directed evolution strategy. So you are evolving the strain in a smart way. In a less time you are actually through engineering technique, you are actually evolving the strain much quicker way so that you can produce your desired product.And also you have seen that the cellular metabolism is a complex network, where the metabolites are interconnected and the reaction enzyme all these are actually very well connected and the computational modeling is required because the production of metabolite is a systems biology problem. How these reactions are interconnected which I have already told you need computational modeling.It is a invaluable step in determining the optimum engineering to reach desired product. Suppose you want to improve the desired product then you have to do some kind of computational modeling to understand the how the carbons are moving or the flux is going from one reaction to the other. And finally, we also learnt what are the application of metabolic engineering and many area metabolic engineering is used where you can see the improved production of certain chemical or byproduct, which is helpful for the society.(Refer Slide Time: 52:34)So these are the references you can learn about the fundamentals of biochemistry by Voet & Voet. And also you can learn about genes by Benjamin Lewin. Also for computational analysis of biochemical systems also you can learn this technique from the book given by Eberhard and Voit. Thank you.