Loading

Module 1: Biotransformation and Genetic Transformation

Notes d'étude
Study Reminders
Support
Text Version

Optimization Strategies

Set your study reminders

We will email you at these times to remind you to study.
  • Monday

    -

    7am

    +

    Tuesday

    -

    7am

    +

    Wednesday

    -

    7am

    +

    Thursday

    -

    7am

    +

    Friday

    -

    7am

    +

    Saturday

    -

    7am

    +

    Sunday

    -

    7am

    +

So, the disadvantages of natural plant extraction, it includes the low concentration of the product in the plant . There may be because of thedemand, supply difference there can be limited supply, it can be an endangered plant becauseof extensive uprooting by the industry . Then it can be an endemic plant not able to growelsewhere only on a particular region , so there can be limited supply for these reasons. Now, endangered species I said can be a limitationwhich may lead to ultimately the extension of the plant . Then natural plant becauseit is seasonal the compound is temporal and spatial variation is there, So, because ifit is seasonal then there is long time required maybe it only comes up when the matured plantcomes up , so non uniform and inconsistent quality and quantity .So, what are the alternative production methods for such high value phytochemicals eithersome as I said we had seen how complex are the structures and biosynthetic pathways . So,these with very high value and low volume metabolites very complex structures too manychiral centres, so they it has been observed that chemical synthesis is very difficult.And even if it is people are able to do it , it becomes a multi step process . So, biotechnologybased methods what can be biotech base methods? One is plant cell based bio process , whatelse ? .Which is not mentioned here. Protein engineering.Protein engineering. .Where means heterologous expression you are talking about , so in some other host systemeukaryotic host system. But, some of the secondary metabolites people are trying the most frequentlyused system is yeast where people being a eukaryotic organism people are trying to express. But the challenge is that some of these high value metabolites or biosynthetic pathwaysnot elucidated. So, now how to get those intermediate steps,the protein engineering which is required you need to know which proteins are involved.Then the other is it is such a wide and big spectrum like for example, 40 or 50 stepsare involved and if it is a heterologous system where the pathways not present. So, all thosesteps have to be included, so it is very challenging which may cause the yields are very less whichcan make the process non economical and challenging .So, the most feasible process which is proposed is which we would like to proposes is plantcell based bio process where the machinery is present. But you need to work around thedifferent other bottlenecks which is which can be the low yields and scale up of theprocess. So, the advantage of producing these highvalue metabolites which are low volume in nature using plant cell technology can be. There can be control of supply why? Because the product is independent of the availabilityof the plant itself and they it is away from climatic, geographical, and governmental,restrictions . Then high growth and productivity, so high growth and productivity as comparedto the natural plant how , high growth and productivity as compared tonatural plants . Productivity because we can culture them inless amount of time. Right.It can optimize the production according to our needs.So, growth rate because these are amenable to optimizations . So, you can end up thetime is the big factor you can run batch to batch some it is months or years where youwill have to wait here you can run a reactor and harvest . So, reduction in time and spaceis logical ; requirement for the production of your desired compounds.Now, strain improvement it is also the cell line itself is amenable to optimisation. So,you can manipulate the cellular yields to improve the y p by x which is product yieldper unit biomass . So, there is ease of extraction and purification why ?Because we are not getting an mixture of products we are getting.Because you are doing process optimisation, so,you will be optimising the conditions soas to increase the productivity of your desired compounds. So, what happens that the relativeabundance of your desired compound becomes more than the other small molecule or otherimpurities, so there by the downstream processing can become easier .So, what do we need for plant cell cultivation we have now studied all this , an appropriateexplant is needed, a suitable growth medium; what does the growth medium composed off.You will have major salts, minor salts, energy sources, growth regulators in it which willfull fill the plant cell growth and this can be what form? Liquid, or semi solids .So, aseptic conditions are required, so as to prevent contamination specially from microbes. Now, optimum culture conditions, so there will be external culture conditions meansenvironmental conditions and other physical and chemical parameters .So, this is a schematic how do we begin? We know we begin from the plant parts then webring it in the form of a de differentiated form which is callus. From that callus youcan select the cell lines, bring it in suspension do the optimisations where yield enhancementstrategies are implemented . So, as to improve the y p by x what kind ofstrategies come in this? Your improvement, your media optimisation , precursor feeding,elicitation, and genetic engineering which can be metabolic engineering . Then come tothe bioreactor stage once a suspension has been optimised at bioreactor stage what canbe done . You again optimise the bioreactor operatingparameters which we also called as fermentation parameters. Then you can optimise the operationmode , mode of cultivation which means can be feeding strategies in order to remove what,why do we have to have feeding strategies, what is the aim , why are we talking aboutmodes of cultivation here every time in the reactors , why or the reactors cannot be runas batch ? .To remove nutrient limitation and nutrient innovation if it is there , so because inorder to improve the productivity your yield yield you would like to increase the biomassor the product. And why in a batch it is getting limited? Because of the nutrient imitation,so nutrients feeding is done to get over this nutrient limitation.And now with the logic that if nutrient limitation you can dump in all the substrate, so thatit is never limited, so then a innovation can play a role . So, there comes the roleof how do you feed, when do you feed. So, then after you have grown the biomass it comesto downstream processing, downstream processing means separation, purification of the product. So, what are the approaches which we are goingto discuss, which can lead to improvement in yield and productivity of the secondarymetabolite, optimisation of media composition, optimization of environmental factors , additionof precursors, cell permeabilization, addition of elicitors , immobilization of plant cells,and high density cultivation in reactors. And in some of these is in order to reducethe hit and trial and minimise the number of experiments which you would like to conductto reach to the optima we will see how mathematical modelling is used with case studies .Now, yesterday if you remember I was talking about that in order to develop start the bioprocessdevelopment you would like to begin with the .Highest. Highest yielding cell line, so logically youwould like to pick up first the plant variety which is highest yielding, how is screeningand selection is it same or is it different . Be it microbial, be it animal, be it plantcell fomentation, these techniques remain the same of optimisation, when we say screeningand selection . Screening.She was close , screening is a passive technique , selection is an active technique . Screeningis what she said for selection in which you screen the cell line based on how would youscreen suppose , any example . .Hm. Like improve by screen culture.Ok, so you will engineer the cells first . For blue white.For blue white .. Mam, there is certain there is certainbacteriawhich canutilize the blood organ some which cannot. So, in this screening process whenwe subjected to this same media, we can understand which are the cells whichutilize the mediaand which are ones which cannot, so, that is the screening process .No, so extrapolated to secondary metabolites from plant cells.. We need to select the highest yielding callusline . So, once.Or the cell line callus is a mix of a number of cells is not it. That is why we say thatcallus would lead to difference in with subsequent sub cultures you may find that the yield keepson varying . Right co culture and see the bacteria is getting. Bacteria where is the bacteria coming?Like say if the plant is producing a particular metaphyte they will defends against the bacteriathat will be kind of a test organism to find out the production is actually happening ornot you can into bacteria to find. On paper this can be done, think in termsof you are given and you need to develop a bioprocess. So, think simple which can costyou less, which can easily be implemented and then you need to take the cell lines.So, once you start growing bacteria along with plant cells plant cells will not survive. Mam, taking our point we can just introducesome amount of stress conditions in which you offer a secondary metabolite is produced.And the subjected to all of those callus lines and we see which of them produces the mostof amount of secondary metabolites. That is correct , but the only check hereis that you are now getting you have opted for a material which is known to produce thatcompound you have a number of cell lines. So, one is you check the yield of the productand you select the highest yielding cell line and the other if suppose it is some of thesecond if it is pigment based secondary metabolite. So, then you can take that piece of calluswhich demonstrates that colour if it is a coloured pigment anthocyanins or any colouredother pigment. Then you know from that mixture of cells which cells will be producing thatcallus or as you people said that if it is now coming on to selection which is you wouldlike to only take the desired cell lines the others you do not care.So, let them die only the ones which you want to select should survive, it will be usefulwhen you are selecting a plant cell line for what for give me some examples for what purpose. If I am trying to find a cell line which is highly resistant , suppose I am lookingfor salinity stress as you are saying, so only those which can resist will survive theothers would not survive . So, which subsequent sub culturing and acclimatizationalso you will end up having cell line which is resistant to the stress and therefore,you can pick up the desired rate cell line in the cell line . So, now plant cell cultureswe know that they are genetically heterogeneous means what, that they change in chromosomal.So, that was what when I say change in chromosomal number, arrangement, what kind of variationam I talking about . .Soma clonal variation, so epigenetic instability, epigenetic instability means change in thegene expression or cellular phenotype caused by mechanism other than changes in their DNAsequence . So, which causes genetic instability leading to product accumulation only in somepopulation of cells that is the problem which the heterogeneous callus would have . Now,productivity in such mixed population of cells is liable to be unstable, so generally whenwe induce from a explant a callus it is not called as a plant cell line.Because from the wound which you create and the cell start around that wound the cellwill start rapidly dividing and forming a de differentiated mass. Now, all these cellshave a different makeup and as you would grow it on a solidified medium they would multiply.So, some of the cells which are close to the medium will have more approach to the nutrientsand they are more the nutrients are more available to those cells than the cells which are atthe top. So, there are biochemical gradients which may form which may lead to ultimatelychange in their biosynthetic potential . So, it is generally observed that when youbegin with callus lines, either you first bring it to suspension and through the cellsewing if you remember I talked about the cell sewing technique. In order to get toa synchronous culture based on the size with an assumption that the cells of same sizeand shape can be assumed to be in the same metabolic state .So, there by leading to a synchronous culture or you keep sub culturing them such that underthe conditions which are preferable for the secondary metabolite of interest such that,during the subculture natural selection happens. So, you will only the preferred the cellswhich are capable of producing under those conditions the secondary metabolite wouldcontinue to grow and later they would be the majority .So, it is always the kind of cells in the callus and the number of those cells in thatcallus which are capable of producing your secondary metabolite. So, how to increasethat number and it is generally observed that when you begin with the callus induction theyields for initial year or so oh you you will see a large variation. Only after 3 4 yearsof sub culture you will find that it comes down to a consistent product yield .So, productivity in such mixed population of cells is liable to be unstable since changein any factor. That is the very reason why we in lab always insist on keeping the conditionsconsistent you cannot or take lightly the sub culturing of the callus the parent materialwhich is going to form your master cell bank. So, under the conditions which you subculture,the time at which you subculture you cannot think that if it is 30 days, let me do itin forty days it does not matter . Everything matters the time at which you are sub culturing,the conditions today you had put point eight tomorrow you had put 0.6 it is going to vary. So, one has to be really careful while subculturing your master cell bank . Since changes in any factory environmental, or nutritionalwhich favours the growth of a particular sub population of cells there by changing thecomposition of the culture the variation in culture .Now, because it is something which can tweak the bio synthetic potential of your cell mass,so it also becomes a tool for variation . So, the variation in the culture offers the opportunityof isolating from the mixed population of variant cells, the ones with high productivecapacity . And thus developing from them cell lines of high productivity .So, the rate of accumulation within the productive cells and the proportion of such cells inthe culture is what which governs the yield or selection of the high yielding cell line. The low production in cell culture can be due to one is competition between the primaryand the secondary pathways , the same intermediates as we have seen the pathways that then bifurcatefor secondary metabolism. So, there will always be a competition ofcarbon flux. Lower expression of key enzymes in the rate limiting steps in the pathwayor gene expression of the secondary metabolism can be a limitation . Then screening is apassive technique by which a number of cells are analysed for the desired rate .So, we generally as you were saying we would look for the highest yielding cell line, wewill do some part of the callus we will take out we will do extraction and we will do yieldin analysis. Or if it is a coloured pigment you can directly view it from your it is visibleto eyes , if if it is if it fluorosis under UV then to you can do it by UV light.So, whether the callus which you have obtained is producing your compound or not you cancheck . Now, selection is what? Selection is an active process by which it deliberatelyfavours only the survival of the desired variant and the wild other type of cells they willdie . So, callus systems they are used for screeningand selection . Cell lines which accumulate high levels of coloured compounds can be selectedthrough visual observation. The callus can be checked visually for altered pigmentationby the naked eye for coloured spots under UV lamp.Screening of pigments for example, if suppose you know from literature that greener is thecallus it is somewhere linked to secondary metabolism of secondary metabolite biosynthesis.So, you would like to select the most green callus line, so screening on the basis ofpigments like chlorophyll, anthocyanins, napthoquinones, carotenoids, these are all coloured pigments.So, if you are looking for such kind of pigments it becomes very easy to do the screening .Now selection can also be performed by using your screening techniques, screening meansmeans using different mesh sizes and trying to reduce the mesh size for example, fromfrom 100 microns to less than 100. So, some suppose 500 greater than 500 microns to lessthan hundred microns . So, the same thing that you can end up ina synchronous culture, the selection can be performed with the fine rapidly growing suspensionconsisting of aggregates of 50 cells or so. Now, fine cell suspension is suitable in selectionfor resistance why do you think every time the stresses on fine cell suspension for selectionprocess , how will it help than the lump culture .So all sensor uniform in a metabolic activity. We are talking in terms of selection , selectionmeans . So, the size variation will not affect theselection process, like all the cells will have the same .That was sell sewing , now in general there can be more than apart from cell sewing methodsdirectly like for example, if you are looking for resistance . So, the rest will get killedonly those which are resistant will survive either this resistant can be solved resistance,or can be against the toxic metabolite or antimetabolite resistance.So, if suppose the other get killed and you need to now the only one survives why theyit is recommended to use dilute cell suspensions . Like you were giving an examples for thebacteria you grow it in a solidified medium such that only the bacteria which can degradethat particular metabolite. Or which is resistant would leave and then what do you do you pickup what you pick up the. Colony.Colony , so there also what is recommended to use.Solution. Dilute solutions you can get to the purposeswhat? Selecting the highest yielding cell line a callus is what? A heterogeneous massof cells . So, therefore, what is preferred you bring it in suspension, make a dilutethen select for a resistant cell line and allow that to grow and multiply it to giveyou a fresh callus using what single cell culture techniques which we have learnt before. So, in the selection program for resistancein cell culture one can detect growth of the resistant population. But because you endup in single cells it will become it will take time for the callus to reappear and forthe culture to again come back to a rapidly growing state. So, therefore it is said thatit will take some 1 or 2 months to come back to to reach to a uniform callus a single cellbased callus line . Now, to avoid inherent problems in the useof callus or cell culture for selection and screening, the plating of cells or protoplaston solid media or mattresses can be preferred. Now, in plating the cells, the first stepis to prepare single cells or small size aggregates for plating .The cells have to be plated at lower densities allowing growth of individual, clearly separatedcolonies that can be isolated and then sub cultured. Unlike microbial cells, culturedplant cells require minimum inoculum density this also we studied that plant cells willbe needing minimum inoculum density for the growth to begin .Generally plants cells are hm plant cells are.. Are grow.Communicate each other. They communicate with each other through.Transferred as parts. So, there is cell to cell contact which isnecessary , there is communication happening, so to facilitate and bring it to that conditionthat minimum inoculum density is needed . Now, preparation of single cells or protoplastwe have already studied to filter successiveleave cells of fine suspension culture through,so we use cell sewing techniques. So, where around 500 to 75 microns you keeppassing the cell suspension this will end up in synchronous culture . The obtained suspensionwill consist of single cells or small aggregates , which most likely have originated from singlecells why how can you assume that . When we are using these cell sewing techniques weare starting from big size and we are ending up at a very small size 75 micron cells areless size . So, generally the cells we said 50's or 100cells together would be there in cell aggregates and the size is around plant cell size thatalso be dead average . If you know that average you will get to the logic why this sewingtechnique of 500 to 75 will end up in either single cells or will end up in very smallaggregates which would; obviously, be coming from the single cells. Because they have multipliedand formed a little bigger than the single cell .So, large aggregates suspension adding pectinase helps how this also we have done that is thevery reason we were doing all that . Generally you will find that even when you will inducea callus from different types of explant they will all vary not only in their biochemicalmakeup they will also sometimes vary in their morphogenetic or morphology.Some will be very hard and compact, some will be very friable that you will just touch witha spatula and they will disperse in the medium. So, that is because of the friability is becauseof the water content in the callus. So, large aggregate suspensions we are using pectinasenow because it is preferred that the callus should be friable in nature . If you wantto generate a suspension, because it can easily disperse in the liquid medium . Screeningand selection should be done from a freshly prepared cell suspension from the plant thanfrom a very old suspension why? .Why should you prefer young? Because they will be more actively growing cells. The mostideal tissue for isolation of single cell is leaf and mesophyll cells which can be mechanicallyand enzymatically isolated . In order to select a single cell for plating cell suspensionsmust be diluted to densities where the plant cells do not grow without special supplementation.So, what are the methods of screening and selection, secondary metabolites it may showan intense colour, so visual detection you can take that piece of the callus and startpropagating that callus. So, you will end up in a callus line which will is giving theproduct of interest . Altered pigmented areas of the callus can be easily detected or directanalysis of the cell extract is done to know the quality of the clone. The clones underinvestigation have to be divided for subculture and chemical analysis .