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Module 1: Biosynthesis and Cellular Transport

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The Cellular Transport Process

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Metabolic Engineering
Prof. Pinaki Sar
Department of Biotechnology
Indian Institute of Technology-Kharagpur
Lecture - 08
Review of Cellular Metabolism - Part C
In today’s lecture, we are going to review the cell metabolism with respect to the cellular transport process.
(Refer Slide Time: 00:36)
(Refer Slide Time: 00:40)
Now cellular transport processes are divided into two main types and subsequently into subtypes. The first one is the free diffusion and this is followed by the facilitated
diffusion and both this free diffusion and facilitated diffusion are considered as passive transport since they do not depend on the supply of free energy like ATP.
And the next one is the active transport which requires input of free energy for example, from the ATP or other energy sources within the cellular systems. So there are certain other types also which are not included in this type of list like they cannot be differentiated between passive or active processes. So we will talk about them in this lecture also.
(Refer Slide Time: 01:52)
Now nutrient uptake by the cell has certain very specific properties. The first and foremost is the specificity of the uptake process. So most of the cellular uptake or cellular transport processes are developed, so as to pick up the necessary molecule only out of the numerous types of molecules there in the outer environment.
As the cellular system has evolved within the natural environment, where the number of molecules present in the outer world could be very high in terms of both the types of the molecule present and their chemical properties and their requirements. So the cellular transport process has been evolved with a very specific nature of transporting the required molecule only inside the cells and other non required molecules may be excluded from the transport process.
The next one is the uptake against concentration gradient. The concentration of the solute or the molecule which is to be transported inside the cells might be very low
outside and compared to the inside of the cell, yet the cell might be requiring that particular solute molecules.
So solute transport would be against the concentration gradient. And otherwise, if the outside concentration is very high and inside concentration of the solute molecule is low, then the transport might be occurring through the concentration gradient and we will be discussing both this type of through the concentration gradient or the down the concentration gradient transport as well as against the concentration gradient transport as well.
But the main point of focus would be that the transport could be against the concentration gradient because in cellular systems, often many of the required metabolites are required solute molecules, nutrient molecules are present in relatively higher concentration inside the cell compared to outside environment. But still, the transport process allows the cell to move the molecule inside the cell only.
The next one is the characteristic property of the membrane through which the transport occurs. So that is nutrient molecules must pass through a selectively permeable membrane. The membrane could be the cellular membrane, it could be the inner membrane or plasma membrane, or it could be the outer membrane in case of gram negative bacteria. So we generally refer these as membrane permeability.
It is the characteristic property of the cellular membrane that will allow only selected molecules to pass through it. Others will not be allowed to pass through freely. So when we say that the some molecules will be allowed to pass freely and some molecules would not be allowed pass freely, we refer to the selective nature of the membrane.
So for the molecules which are generally not allowed to pass through freely, for them cellular system has evolved certain mechanisms by which such molecules, which are not allowed by the membrane under normal circumstances would be made available inside the cell. So we will be discussing briefly about those processes as well.
(Refer Slide Time: 05:42)
Now here, let us look at the cellular structure particularly the cell membrane structure. This will be essential for us to understand the membrane transport process and cellular acquisition of different metabolites including the nutrients etc. So one of the most important components within the membrane is the phospholipid bilayer.
(Refer Slide Time: 06:04)
Now this phospholipid bilayer is also referred as a lipid bilayer because, characteristically it is having two layers of the lipid molecules and these two layers of phospholipid molecules because they have a phosphate group attached to it, it provides a kind of both hydrophilic and hydrophobic nature of the membrane structure.
It allows the small molecules such as water, oxygen and carbon, particularly the molecules which are having molecular mass less than 600 Dalton or so to enter or leave the cell easily. Now if we look carefully within this membrane bilayer structure, the two outer surfaces, this surface and the surface over here is considered as hydrophilic because they are having the groups which are like phosphate groups and the glycerol moieties which are available for interaction with the water readily.
Whereas, the internal portion of the membrane which is composed of mainly the lipid molecules which are representing the hydrophobic part of the membrane. So the lipid bilayer structure represents both the hydrophilic part and hydrophobic part.
(Refer Slide Time: 07:34)
Now in association with the lipid membranes or the bilayer structure, there are protein molecules present within the membrane. These proteins can be found within a surface of the membrane like they may be bound to the surface either the inner surface or the outer surface or they may be across the membranes which are called transmembrane proteins.
These proteins are involved in a number of processes which are related to the cellular transport and other essential functions like the transportation of different molecules, as we will see. They are involved in often different enzymatic functions. They are also involved in as a messenger to send messages to other cells and as a cell recognition system. Thereby, they help the cells to recognize the invader molecules or harmful cells or molecules and also provide supports to the cellular membrane structure.
(Refer Slide Time: 08:30)
So there are also carbohydrate groups attached to the membrane like the glycolipids or glycoproteins, which are found on the surface of the cell membrane and made up of carbohydrate plus lipids or carbohydrate plus proteins. And the primary function of these carbohydrate moieties or carbohydrate chains of different kinds is to recognize the harmful cells. So basically they help in the cell-cell recognition system.
(Refer Slide Time: 08:57)
So now, we move on to the first type of transport which is called the passive transport. So we mentioned earlier that there could be two major types and there are actually subtypes. So total three types of transport we mentioned in the initial discussion. So the first one is the passive transport which will be having two specific type.
One is the simple diffusion type and other is the facilitated transport or we call facilitated diffusion also. So the broad characteristics of the passive transport are that it relies on diffusion only. That means, from a higher concentration to the lower concentration regime, the transport of the molecules or the transport of the solutes occur. It uses no energy because it is down the concentration gradient, the energy requirement is not there.
(Refer Slide Time: 09:55)
Now let us look at the processes involved in this passive or simple diffusion, which is basically the random movement of the molecules, which allow them to spread out or diffuse out from an area of high concentration to an area of low concentration. Like if you have a higher concentration outside and the lower concentration inside these molecules will tend to diffuse.
And if they are allowed by the membrane like they are soluble in the membrane or membrane permeability is there for these molecules, the molecules may move inside the cell unless and until the equilibrium is attained. And it basically involves three steps. So step number one is the transport of the compound from the extracellular medium to the membrane phase.
So so the first one was the transfer of the compound from the extracellular medium like the extracellular medium is over here, from that to the membrane phase, which is the hydrophobic phase. So from the hydrophilic site to the hydrophobic phase, that is the first step.
The second step is the diffusion of the compound through the lipid bilayer like through the hydrophobic zone, which is within the membrane itself, membrane bilayer itself, the movement of the molecule occurs, that is step two. And the step three is the transfer of the compound from the membrane phase that is the hydrophobic phase to the cytosolic phase.
So we could identify easily three phases within this passive or simple diffusion across the lipid bilayer, which is the step 1, step 2, and step 3.
(Refer Slide Time: 11:33)
Now ideally, these steps are well marked over here, the step 1, step 2, and step 3.
(Refer Slide Time: 11:41)
Now this concentration of proton which is building up is actually developing the proton motive force, okay. And this actually disequilibrium or inequality in concentration of the protons or it may be sodium ions sometimes, so that the disequilibrium of these ions okay causes a kind of a pressure to the membrane and that is generally referred as the proton motive force.
Now this proton motive force enables the either to drive some kind of pump or functional units to utilize these disequilibrium to their favor. Now this as we have briefly discussed that this could be connected to a ATPS, ATP producing enzyme system that the protons will be allowed to move in and ATP will be produced.
Now this ATP, which is produced can be utilized to transport a particular molecule like in this case, the solute molecule that was we were referring to earlier. So the
solute molecule can be transported by utilizing the ATP molecule which is produced by utilizing the proton motive force. And this is considered or called as the primary active transport.
On the other hand, the proton motive force can directly be used to transport some other molecule like galactose or lactose etc., which is directly related to the proton transport. Because the proton will be transported down the concentration gradient because it is outside high concentration inside low concentration.
So proton transport will be down the concentration gradient, but the lactose or galactose or similar other solute molecules, which are waiting to be transported inside the cell might be transported through those type of transporters. So now, I must mention over here this the type of the transporters are very specific.
Whenever we are using a ATP based transporters, which are involved in primary active transport, so there are entirely different set of transporter molecules, because lot of conformational change and ATP hydrolysis and all these events are included. So there are specific set of transporter proteins which are involved in primary active transport and also specific set of protein molecules or carrier protein molecules are there which are involved in the secondary active transport process.
So for each of the type of the molecules or each of the type of the metabolites or solutes to be transported, and whether ATP will be utilized or whether proton motive force or a ion motive force will be utilized, based on that all these carrier transports transporters are produced by the cells and cell controls the synthesis of all these carrier molecules considering as we mentioned earlier, the availability, the requirement and the concentration of all these molecules.
(Refer Slide Time: 36:23)
Now active transport mediated mediates the entry of virtually all other nutrients and the mechanism however, may vary. So there might be two or three broad mechanisms by which the active transports occur. Now all of these different mechanisms by whether it is utilizing ATP hydrolysis or it is utilizing the iron motive force or proton motive force, everyone is using energy to pump molecules into the cell at a very high rate and it is against the concentration gradient.
So there are, these are the two important aspect of this transport, active transport that they transport the molecules at very high rate because the carrier molecules are very efficient and once they are present and they are able to recognize the solute molecules they will be transporting the molecules in a very high rate. And of course, against the concentration gradient.
Sometimes even it is several orders of magnitude the concentration difference might be there. So from so they can still transport very efficiently. And nutrients are concentrated inside the cell even up to 1000 folds. So within the cell high and high concentration will be built up compared to the outside environment.
But the still the cell will try to acquire the nutrients because maybe the cell is under the impression that the demand for the metabolism is so that those kind of nutrients will be readily metabolized like the galactose or lactose etc. The carbon and energy sources are so demanding the cell would always like to take them up and inside the cell they would like to utilize them.
(Refer Slide Time: 38:01)
Now the mechanisms of active transport. So we will be discussing two main mechanisms first. The first one is the ion coupled transport, which is considered to be a preload system. So it is called ion coupled transport, because it is driven by electrochemical gradient. We were referring to this earlier, that is either a proton gradient or a sodium gradient is established across the membrane.
And it is a kind of a prior requirement that this gradient is established. Since we are talking about a preexisting membrane gradient or gradient which is first established, it is called a preload system. So because if we want to transport a particular solute through ion coupled transport, first we need to establish this electrochemical gradient by transporting proton or sodium.
Now this gradient of sodium or proton can be created by electron transport, which is coupled to the electron transport system or oxidative phosphorylation or by ATP hydrolysis bound ATPases. So there are actually a number of transport systems, active ion coupled systems are there where ATP hydrolysis is used to transport sodium for example, outside the cell, so that a sodium gradient is created.
And then the sodium gradient will be utilized to transport the actual or the targeted metabolites. So the primary event is the membrane is energized. Membrane is first energized through electron transport based or ATP hydrolysis based creation of the
gradient, gradient of proton or sodium. And the secondary event is this energy is used for the transport.
Now once the gradient is established, this gradient is utilized for the transport to happen. Now there are two important characteristic properties of this kind of ion coupled transport system. The one is the transport occurs in either direction. So it can be cytosolic side to the outer environment or from the outside environment to the cytosolic site and it can translocate either one solute at a time that is called uniport.
Or it can transport two solute molecules, either in the same direction is called symport or in the opposite direction that is called the antiport system. It is very common among the aerobic microorganisms, aerobic organisms also which can generate an ion motive force more easily than anaerobic system because you need to spend lot of energy to generate this gradient across the membrane.
(Refer Slide Time: 40:31)
The next one or the second one is the ATP binding cassette transport or ABC transport. ABC transport as it says it involves ATP hydrolysis, and it uses the energy from the hydrolysis of ATP to pump the solutes. It is more like a direct event. It involves the specific binding proteins located on the periplasm.
So it has multiple steps. So let us just go through the brief steps that it has some binding proteins located on the periplasm, particularly in the Gram negative bacteria
or attached to the outer membrane of Gram positive bacteria that confer the specificity of the transport process.
(Refer Slide Time: 41:09)
So here is the membrane and here are the carrier transmembrane carrier proteins, which are called ATP binding cassette. So these are referred to as ABC transporter. So entire setup is ABC transporter. It has three components. So component number one is this protein molecule, which is a kind of extra to the membrane. Either it is present in the periplasm or maybe on the outside of the outer membrane.
So it is responsible for binding the substrate very specifically and then bringing this whole complex to the transmembrane carrier complex. So that the molecules of solute molecule can be available for the transport. The one of the binding, this protein carrier protein complex is also having a ATP binding site where the ATP molecules are able to bind and hydrolyze and thereby providing energy to the system.
Now let us see how ATP hydrolysis can trigger the opening of this membrane. Now as soon as this binds to the binding protein, which is an extraneous component of the system, so these enable the particular solute to close or available to the carrier complex or the carrier molecules and then the hydrolysis of ATP occurs. As soon as the ATP hydrolysis occurs, the membrane channel gets open and the solute molecule is able to transport within the membrane.
And then this binding molecule is again free and it is able to bind another molecule and then the same way the process goes on. Now without the ATP hydrolysis, this channel will be remain closed or the transport occurs only in one direction. Because these are highly specific transporter, because the entire system is dependent on both the three factors.
One is the binding molecule and other is the ATP hydrolysis component and third one and very important one is the transmembrane domain. So since all these components are highly organized, the direction of the transport is highly specific. It is unidirectional only. Extremely common among bacteria. Nearly half of the substrates transported in E. coli are through these ABC transporters.
So ABC transporters are extremely popular within the microbial system.
(Refer Slide Time: 43:30)
The next one is the group translocation, which is a very interesting transport process, which is also called as phosphoenolpyruvate sugar phosphotransferase system or PTS system. So the involvement of a phosphoenolpyruvate, one of the important metabolites within the glycolysis or Embden-Meyerhof-Parnas pathway is present in this transport system.
The phosphotransferase enzyme system transport a solute like a glucose or mannitol or some kind of carbon substrate like this, while simultaneously phosphorylating it. So the point of interest over here is the molecule, sugar molecule is transported inside
the cell. But as soon as it is transported inside the cell, it is phosphorylated. It is very common in bacteria and it is responsible for acquisition of a number of sugar.
(Refer Slide Time: 44:20)
So let us see how it works. So it has actually multiple components as we can see. Here are the phosphoenolpyruvate, which is hydrolyzed and releasing the phosphate group, activating some enzyme complex like E1, then HPr. And then the phosphate group is transferred to these other complexes. So we are not going into the detail of the complexes.
But the main point over here is that cytosolic hydrolysis of the phosphoenolpyruvate and transfer of the phosphate group is connected to a series of protein complexes and eventually the carrier molecule is activated. As soon as the carrier molecule is activated the glucose or mannitol or this kind of sugar molecules are allowed to be transported inside the cell.
That means the channel is open only when it is activated and the activation of this particular last protein complex, which is a transmembrane domain is achieved only when the associated cytosolic site or counterpart of the protein complex is activated through the phosphorylation. Now the energy is expended not for the transport process directly, but to form an intracellular derivative.
So here derivative means we see that glucose is transported and converted to glucose 6-phosphate. Mannitol is converted to mannitol 1-phosphate. So the energy which is
available here like phosphoenolpyruvate is given finally to the carrier, but the carrier eventually transfer it to the solute molecules which is entering like glucose is converted to glucose 6-phosphate.
Now this is called intracellular derivative of the solute molecule or the molecule which is transported inside the cell. The sugars are mostly the candidate molecules. Now this intracellular derivative, so mannitol is converted to mannitol phosphate or glucose is converted to glucose 6-phosphate, this intracellular derivative is membrane impermeable and trapped within the cell.
So one very interesting strategy developed by the cell is the cell will continue taking up glucose and once the glucose is coming inside it is converted to 6-phosphate and this is, the 6-phosphate molecule is unable to leave it. So they are considered to be trapped inside the cytosol unless and until the cell is going to utilize them. Now phosphorylation also makes the transport of glucose by the PTS free energetically.
So ideally it is considered to be energetic energetically free and it is like two for one deal. So how about two for one deal? Like we are spending one energy over here in the form of phosphoenolpyruvate, but we are getting two things done. One we are getting these transport done which is which may be against the concentration gradient also or the second one is we are converting glucose to glucose 6-phosphate.
Now what is benefit in glucose to glucose 6-phosphate that glucose to glucose 6-phosphate conversion is a very important energy consuming step otherwise in glycolytic reaction or Embden-Meyerhof-Parnas pathway or even the pentose phosphate pathway. Everywhere we see the transported cytosolic glucose is converted to a glucose phosphate molecule.
So that phosphorylation otherwise would require an ATP molecule. So that is the ATP molecule which is now would not be required, because the group translocation allowed the formation of a phosphorylated glucose itself within the cytoplasm. So that is why it is considered as a two for one deal. So by spending one ATP or ATP equivalent high energy phosphate compound, we are gaining two functions done.
That is one is a transport, another is the first step of the glycolytic reaction is completed.
(Refer Slide Time: 47:55)
Now let us briefly discuss or talk about or give some example of understanding the metabolic metabolite transport, its importance in the metabolic engineering or strain development because it has been considered to be giving an upper hand in microbial strain development or microbial cell factories.
Now there is a nice introductory remark about this that what if we knew how metabolites were transported inside and outside the cell. What we can do if we know that how the metabolites are transported inside and outside the cell, like the details of the process. Like how the cells are exporting or importing the molecules would help us to improve the process performance and lower the cost of downstream processing. So we will look at some of the examples.
(Refer Slide Time: 48:44)
So these are called actually transporter engineering in terms of the cell factory production or the advanced level of metabolic engineering and transporters that import substrates can increase the substrate uptake rates and hence increase the volumetric productivity. So enhanced transport of metabolites or substrates inside the cell.
Now the uptake of transport or uptake of the molecule or transport of the molecule which are substrate molecule for the entire biochemical reactions are understood as they will be able to channelize more flux towards the desired reaction. But we can do this thing by multiple ways. So one is utilizing the existing and efficient transporters like the glucose transporters, which are more efficient and allowing more glucose to be transported inside the cell.
But at the same time, using some unique transporter like GatA, which is capable of transporting galacturonate inside the cell. Now the galacturonate is a very important component coming out of the kind of pectin residues present in the plant biomass.
And if we are able to engineer our cells during the metabolic engineering program, the galacturonate which is entered inside the cell through this type of specific transporter can be processed towards formation of meso-galactaric acid and meso-galactaric acid has found to be very important advanced biotechnological applications.
(Refer Slide Time: 50:17)
Similarly, the use of xylose a pentose sugar which is again present in abundance within the plant derived biomass into ethanol production. Engineering a xylose transporter like Gal2 and with a xylose isomerase keeps the internalized or cytoplasmic xylose close to the xylose isomerase and avoiding its use in other pathway.
So the internal of cytosolic xylose can be converted to xylulose and then xylulose will be eventually converted to ethanol very effectively.
(Refer Slide Time: 50:50)
There are also cases where we see that there are leakage within the metabolic reactions. So particularly in multi-step biosynthetic pathways like lysine production or anti the amino acid production, there are problems with leakage of some of the
intermediates. Like as you can see here, the cadaverine or the aminovalerate, these are commonly found to be leaking from the cellular environment.
So lowering the flux towards the desirable the production compound, okay, like in this case from towards the glutarate production or so sometimes we see that the leakage the production is severely disturbed. So the engineering the particular transporters for the cadaverine or for the aminovalerate like the GabP or PotE for cadaverine helped the metabolic help in the metabolic engineering towards the improved performance of these biochemical reactions, because the leakage of the substrates or intermediates are stopped.
(Refer Slide Time: 51:51)
So similarly, within the cell there could be some toxic products produced and these toxic products need to be removed from the cell like in the fatty acid biosynthesis, we often see there are certain aldehydes produced. So engineering the cells with the transporters which are specific to the fatty alcohol transporter make transporting these fatty alcohols from cytosol to the outside environment.
Or the efficient transport of lysine which is produced inside the cell to the outside the cell which is highly desirable because in lysine producing strain, if we can readily take out the lysine then otherwise, the lysine might act negatively because lysine production lysine biosynthesis is very tightly regulated.
If we have accumulation of lysine inside the cell cytoplasm, then feedback inhibition would implement and the production of the lysine or the reaction towards lysine synthesis would be inhibited. So by rapidly efluxing or exporting lysine from inside the cell to the outside actually helped the metabolic engineers to efficiently manage the entire set of problems.
(Refer Slide Time: 53:03)
(Refer Slide Time: 53:06)
So after talking of all these things, so we will be concluding today’s class and for today’s class, we have used the following references, the metabolic engineering textbook, along with this the Prescott microbiology and an interesting review on transporter engineering and the Microbe second edition.
(Refer Slide Time: 53:25)
So to summarize today’s lecture, the importance of cellular transport in microbial cell factory or advanced metabolic engineering is emphasized. The passive transport, simple diffusion and facilitated diffusion, active transport, group translocation and PTS system are all mentioned. Thank you.