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Module 1: Biotransformation and Genetic Transformation

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Statistical Medium Optimization

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We were talking about pH effect of inoculums, then we spoke about medium. Now, we all knowwhat are the medium nutrients which are required for plant cells we have done it in the earlierclasses. Then similarly we have learnt about different phytohormones which are requiredfor in vitro cultures. Now for example, your cytokinins and your auxins they are generallyused for plant cell cultures because they help in cell division and cell elongation.So,this in turn is related to secondary metabolism secondary metabolite bio synthesis. So, thereforeits optimization might be necessary under in vitro conditions. Other hormones are regulatorslike ethylene or gibberellic acid orabscisic acid; so these areother growth regulatorswhich may play a role in either signal transduction; which is related to intern to secondary metabolisminduction or they are directlybeinginducing secondary metabolite bio synthesis. So, therefore,exogenous addition of such hormones can be connected to yield enhancements of secondarymetabolites in plant cells. When I say yield enhancement I mean y p by x.So, macro and micro nutrients you people all ready know nitrogen generally in the formof as organic sources if you take; then casein hydrolysate and peptone can be used to reducethe production cost. But there one has to judiciously see or select that batch to batchvariation should be avoided in the media or in the result because of the variation inthe concentrations. Further, amino acidsgenerally has nitrogen source only aspartic acid isused not other amino acids and moreover it enhances the cost of production; so, thathas to be taken into account. Then nitrate toammonium ratio or ammoniumto nitrate ratio is found to be crucial in plant cells. If you remember I gave you anexample in in vitro cells like for example,when azadirachtin production if you see literaturewhen it was done using a azadirachta indica plant cells; it was observed that at nitrateto ammoniumammonium to nitrate ratio when is minimized, where evencompletely takingaway ammonium as nitrogen source and only providing nitrate at nitrogen source couldenhance the secondarythe azadirachtin in bio synthesis.So, similarly this gives an indication that the kind of salts or sources which you areusing as nitrogen source will also impact your secondary metabolite bio synthesis; apartfrom the concentration optimization. Now, talking about carbons being plant cells;so if it is a photoautotrophic cultures then you need to provide light and CO 2 is usedas carbon source. But if it is a heterotrophic cultures then we utilize carbon sources likesucrose, glucose. Generally sucrose is preferred because sucroseis a economical carbon source , but if it is a mixotrophic cultures. So, now if supposeyour secondary metabolite is found to be connected with chlorophyll bio synthesis. So, then youwould like to have green cultures. So, for more chloroplast production you might be leadingto incident light on the cultures. So, then gradually the cultures they will become mixotrophicbecause you are also providing a carbon source and there is light in the culture; so thesecultures are called as mixotrophic cultures. So, the possible mode of actions of sucrosewhich are known in literature on cell cultures this includes inhibition of endogenous auxinbio synthesis. By manipulating sucrose concentrations this may indirectly impact the endogenousthe oxygen; auxin levels also. Then influence on differentiation characterized by increasedactivity of the enzymes in the pentose phosphate pathway.So, when I say these as examples to you people; you one cannot generalized it depends on thespecies and the kind of secondary metabolite you are working with; that is why one hasto take cues from literature and then optimize according to the type of culture, type ofplant species and the class of secondary metabolites which you are interested in. So, then we spokeabout statistical media optimization; I will just quickly go through the .So, conventional fermentations we optimize using single factor the rest of the componentsare kept constant and one component is varied in a range and you see the effect on the response.Now, this approach is what? It is time consuming, it assumes that the process variables do notinteract and the process response is a function of single parameter; now which is varyingin a range. Now, statistical optimization methods theytake into account the interaction of the variables as we know in generating the response. NowPlackett Burman design is a well established and widely used statistical technique whichis called as a screening design; which is the two level design in which every factoris varied at a small at a minimum level and at a higher range which is called as minusand plus range and we ignore; however, the interaction among the variables in this design.Now, as I said it istwo factorial design what do you mean by two factorial design? A seriesof runs in which combination of two factor levels are included and offers the screeningof a large number. So, the advantage of Plackett Burman is thatif you have a large set of parameters; suppose 10, 12, 15, 16; then if you have N parametersyou can carry out the design and the analysis with N plus 1 experiments only.It does not describe the limitation it does not describe the interaction among factorsand is used only to screen and evaluate important factors influencing the response. So, whichmeans it will help you in ranking the parameters; it is a resolution three design what doesit mean? It confounds main effects with two factor interactions.So, which means that as I was talking yesterday that it may confound the effect of two factorinteractions. If you are taking two factors then the interactive effect might be confoundedby the main effect of any one of those. So, according to Plackett Burman; it works ontwo assumptions one is called as factors sparsity and the other is called as effect heredity.Factors sparsity means the factors which you have chosen it assumes that they are independentparameters. The second that the interaction for any interaction to be significant on theresponse at least one of the main factors should be significant; isn't it? So, basedon this it fits the response and the concentrations of the factor values in a linear model whichwas linear means taking into account only the main effects.So, then coming on to optimization tools after screening designs; the most widely used toolis response surface methodology. Now, when I am talking about Plackett Burman and responsesurface methodology, it does not mean that these are the only tools available in designof experiments, there are many other design of experiments which can be used.But depending on the assumptions which you can take how how nicely or what factors youwould like to rank, depending on whether the assumptions of effect heredity or factorssparsity can really practically hold through; you must adopt or adapt any kind of such tools.You must look into what assumptions are behind that model or behind that tool and then accordinglyadapt that tool. Now, process optimization tool is responsesurface methodology; it is most often used to determine the optimum response for a specificrange of variables. Now, the interaction of possible influencingparameters can be evaluated in a limited number of planned experiments. So, yesterday I wastalking to you that every factor if it is varied in a range; there can be N number ofpermutations and combinations depending on even between 1 and 2; there can be 1.1, 1.2,1.3, 1.12, 1.13; so there can be N number of permutation and combinations. So, theseare fraction factorial designs which will pick and choose only a fraction of this designspace. And will give you a recipe of experimentsand will be clubbed with statistical analysis in order to make sure that the data fittingwhich is done in a polynomial equation is statistically valid or not. Now, yesterdayI did not talk about this any model will beconfident and you can easily use it for prediction forthat extrapolation of that model apart from the designs page which you have created iscrucial. So, tools like response surface methodologythey will; the Anova which is done or the confidence which is calculated is based on;you will always see that such design tools will always give you some of the recipes,experimental recipes which are lying outside the design space. Any model is said to berobust or applicable only when it is able to predict outside the design space. A modelis a model only when it does not mean you cannot prove a model to be nice using thesame data which you have used to fit the model. Obviously, it will fit the model because youhave use the same data to make the model. So, the fitting of the model of the confidenceof the model is only true when it is able to predict something out of that design space.So, that is what these tools also do; apart from the range which you provide as a userminus 1 and plus 1; there will be some of the design points which will be lying outsidethis design space and the experimental recipe then you will carry out; the analysis whichis done to calculate the confidence or the predictability of the model is based on theresponse which you get in some of the recipes which are outside the design space.So, RSM may be summarized as a collection of experimental strategies or mathematicalmethods and statistical inference for exploring the functional relationship between a responsevalue and set of design variables; so therefore it ispolynomial. Now a central composite designwhich is one of the tools in RSM is usually used to acquire data that will fit an empiricalpolynomial model; empirical means there is no scientific bases to it .A central composite experimental designed coupled with a polynomial model is a powerfulcombination that usually provides an adequate representation of most continuous surfacesover a relatively broad factor domain. Now, talking about precursor addition; weknow what is the precursor, precursor is generallycompound which is an intermediate in or at the beginningof the secondary metabolite bio synthetic pathway and therefore, stands a good chanceof improving the production of the downstream product.Now, any there are different classes of precursors; they are classed as endogenous, exogenous;then they can be classed asindirect direct they are classed as natural then or obligatoryintermediates know what does that mean. Any compound whether endogenous or exogenousthat can be converted by an organism or a living system into the secondary metaboliteof interest is known as precursor. Intermediate compound is one which is both formed and furtherconverted by the organism under identical conditions which means that an intermediatecompound will be formed and at the same time will be catabolized to form your product.So, intermediates can be classified as natural intermediates; a compound formed by the organism,independently from the investigated bio synthetic pathway. Obligate intermediates a member ofthe path although it is formed inside, but it is not directly participating in the biosynthetic pathway. Obligatory intermediate a member of the path using which an organismcan synthesize a given product from the given source material; so it is present inside asthe intermediate. So, precursor feeding can be used to improve the yield of the secondarymetabolite. Now, suppose we add exogenously; now indirectprecursors or I was talking about exogenous precursors which means that anything whichyou add the cell can take up and then further metabolize it to produce the product. Now,why do you think adding a precursors? Sometimes it is seen that you add a precursor exogenously;although you know that it is a part of the bio synthetic pathway and ideally if you addit; it should write the reaction forward, but sometimes it does not happen.So, the reasons which are said to be responsible why despite adding precursors exogenously;there is no response by the cell in terms of yield enhancement, it is the lack of uptake;the cell is not able to exogenously take up the precursors precipitation of the precursorswhich means availability of the precursor to the cell can be a limitation; diversioninto alternate pathways. When added and exogenously added and takenout up up it may get utilized in different other pathways because depending on whichposition it lies because these are all branched pathways. So, and there is compartmentationthere is sometimesthe entire bio synthetic pathway has been divided into different organellsis; there is a transport of precursors which is taking place.So, then if you provide the precursor from outside; this may get diverted towards anyother path way. Then lack of despite be you adding the precursors; what else can be alimitation? Maybethere is not enough enzyme which is available for the precursor to beutilized in that reaction. So, the expression level of the enzyme may be a limitation orthe activity of the enzyme which internally it is expression based .Now, addition of the precursors when I say lack of uptake; so how is it facilitated becauseit has to cross the cell membrane barrier; it may be a toxic metabolite to the cell whenit is present outside. The cell is able to overcome the toxicity you remember I was talkingabout anti metabolites, where I gave an example of biotin. Pimelic acid when being a precursorof biotin and the bio synthetic pathway; pimelic acid is toxic to the cell.So; what it does? What mechanism has happened? May be it is a detoxification process thatthe as soon as the pimelic acid is formed; it is transform to biotin, biotin is a productof interest. So, if you add pimelic acid outside then it is seen that it is toxic to the cell.So, there it will not work or sometimes cyclodextrins are used in which the precursor is the embeddedsuch that it can facilitate the uptake of the precursor.The toxic precursor inside the cell because it will have the same hydrophilic hydrophobicends; it will facilitate the up take through the phospholipid plasma membrane or the cellwall. So, it may take up depending on the transport proteins; depending on the effecton the cell membrane, plasma membrane sometimes in order to facilitate the uptake.So, some of the precursor in some cases they are there which are provided as a complex;they are provided as a complex such that they can be taken in by the cell once in then itcan be utilized . Now addition of precursors for enhancement on secondary metabolite production;it is influenced by which factors? Spatial orientation of the enzymes, then compartmentationof the enzymes, presence of other substrates, reservoir sides for production and accumulationso because of the compartmentation; all this will impact the effect of a precursor exogenouslyadded in order to improve the product yield. Now two methods of increasing the precursorsupply within the cells by adding it into the medium in which case what becomes limiting?The uptake mechanism becomes the limiting factor. By selecting for resistance to precursoranalogous; what does this mean? We have been talking this; by selecting for resistanceto precursor analogues in which case the intra cellular levels can be modified. How? Letus take the example of pimelic acid which I gave its a precursor of biotin.So, been toxic to the cell in order to select a high biotin yielding cell line; people addpimelic acid and try to acclimatized the cell or screen or select the cell it is a invasiveprocess now. So, they will select the cells which are able to survive under high concentrationof pimelic acid exogenously added in the medium. How will that help in selection? Because onlythe cells which are able to take up and detoxify it to biotin will be able to survive and becausethey are able to convert convert them into biotin; they will be?Higher. Higher yielding cell lines of biotin; so,that is the effect of adding precursor analogues and selection process.Now, as an example this is the mevanolate path way through which azadirachtin is produced.So, the red ones are in literature some of the intermediates which have been added exogenouslyto enhance the production of azadirachtin. So, this is a part of the biosynthetic pathwaywhich finally, then leads to azadirachtins at the bottom. Now, the red ones I said thathave been used in literature for production of enhance yield of azadirachtin. Now, acetateah; sodium acetate salt if you provide it in large amounts or concentrations these arealso known as elicitors in literature. You will find that they are also stress enhancingcompounds to the cell. Now, how would you determine that anythingwhich you are adding is a precursor? How will you find out how what kind of design whatkind of an experiments would you design to find out if what you added can be a precursor;indirect direct and not an elicitor. Precursor it will be taken up that whether it is actingas a elicitor or a precursor; if it is an elicitor will it be taken up by the cell?No, if it is a precursor it will be taken up by the cell .So, now coming on to cell permeability enhancers; cell permeability enhancers they reversiblychange the permeability of the cell. So, when I say reversibly what does that mean? Howcan anything in change reversibly the cell membrane permeability? Either it should beable to disrupt or it should not be able to disrupt. Reversibly means what? You do notthink about it; cell permeability enhancer means I am able to open such that things cango in; things can come out, but the many time I am able to open how is it reversible?Sometime people againcome back to the same place.How? .Yes, that is true reversible when we say which means that we have exposed it to a limit fora time such that the damage is reversible; it does not become permanent. Now, what causesthis damage to become reversible is depending on the exposure time, depending on the concentration,depending on what chemical or technique you are using to create these transient pores.Now, there are well defineddefense mechanisms in place in the cell to overcome these transientpores. These transient pores can be caused by date to day damage to the cell membraneisn't it? May be because of the change in the cell fluidity because of the environmentalfactors; there can be a change in the cell permeability membrane permeability or becauseof electrical shock, heat shock that is what you do; there can be pores formation.So, there are more than one techniques in the cell differ sometimeswhich involveah yourthesemembraneproteins small small portions; now these are called as caveats now they are used to sealthese damages. So, it depends this is another kind of defense mechanism which is in placeuse of vesiclesendocytosis, exocytosis which is used to seal up back the membrane. Butif it becomes and because of the tension created phospholipid membranes now they themselvesremained; it is not that they are sued together. Now because of the hydrophobic and the hydrophilicends hence it is intact; any disturbance can cause transient pores.So, there is a tension the minute there is a transient pore there is tension among thesephospholipid bi layers. This tension forces them to come back; only if even despite thefact that there is tension they want to come back and they are not able to it becomes permanentotherwise these are like elastic membranes .So, what kind of permeability enhancer? This is what we utilize under in vitro conditionsin order to improve mass transfer; generally for the uptake of nutrients sometimes youwould like to drive the product formation reaction forward by use of raisins and thiscell permeability enhancers so as to bring the product out from the cell and drive theintracellular concentrations of the product inside. Then they are also used for makingthe process continuous so that continuously you keep growing and the product is comingout. The intra cellular product is coming out in the medium and it is getting collectedso that it there is improvement in productivity of the plant cell bioprocess.So, it will make the process continuous. So, what are the different kinds of permeabilityenhancers which are used? It includes ultrasonification, electroporation, use of surfactants or organicsolvents like n-hexadecane or isopropanol or tritin x; these are some of theliteraturebased or you will come across in literature where these kind of chemicals have been usedas permeability enhancers. But the key there is that the concentrationin which you are using because even if you use surfactants. Now surfactants how do theyincrease; the cell permeability? If you see the use of organic solvents some of them aresurfactants. Solubilize .Right solubilized because they also have hydrophobic and hydrophilicans; so therefore, they willmake transient pores. Now what is crucial here is that the concentration in which youuse it and the time of exposure. So, therefore, when you are optimizing one has to optimizethe time of adhesion because that will determine from the time of harvest; the exposure timeand the concentration in which you are using it. Talking about elicitors we now know whatare elicitors; elicitors these are defined as molecules that stimulate defense or stressrelated responses in plants which result in improve biosynthesis of secondary metabolites.So, now this entire thing will include all that we have studied earlier in secondarymetabolism. This will this can include your PR proteins themselves, this can include yoursignalingmolecules, this can include your pathogenic components, this can include your endogenouslyproduced cell wall of plant cells or any other protein or defense related component whichis produced by the plants cells. So, exogenous addition of elicitor molecules; now they canbe biotic, abiotic depending on the origin; they can beendogenous, exogenous.So, when I say biotic and abiotic; what does it mean? Biotic elicitors would be can youmake out from the word? microbes biological microns.So, which are which have a biological origin; abiotic which are chemical for example, canyou give me an example; abiotic elicitor like? Calcium.A biotic elicitor like? Light.Calcium signaling is a part of ah, but it is also one of the medium nutrients. So, elicitorwould be which can induce the participation of calcium signaling hm. So, abiotic elicitorswhat can be? These are all stress enhancers. Temperature; temperature.Ok. Salts .Salts, heavy metals, then? Biotic elicitors can be fungal components, their cell wallcomponents, chitin. So, elicitor induced effects in plant cellsinclude what? They will they can induce calcium metabolism. Now, calcium metabolism has acrucial; calcium cmp pathway; now it has a crucial role to play in inducing certain proteinsmean and in transcription of certain enzymes which may be involved in the.Yeah. Desired secondary metabolic path way.So, calcium metabolism it is used as a second messenger in the cells; now second messengerit is involved in many signal transduction pathways in plants. So, generally what happensthere can be an elicitor molecule which willcome and bind to the receptor on the cell membrane.Ultimately, giving rise to certain kind ofproteins which will thenah travel l to endoplasmicreticulum and there through endoplasmic reticulum; it will open up the calcium channels. Thesecalciums then the intracellular concentration of the calcium ions will increase and thesecalcium ions will then bind to certain proteins which can act as inducers or repressors inyour secondary metabolic pathways. So, I am just giving you it in short; so thisis how it can get induced, so calcium metabolism cyclic AMPs involved in that. So massive variationin membrane integrities, protein and phosphate metabolism, ethylene production, peroxidaseactivities; now these are the different ways in which an elicitor impacts the secondarymetabolism inside the cell; it can give rise to more reactive oxygen species which meansperoxides metabolism. It can give rise to signaling molecules likeethylene which we have already learnt now in the signalingyour secondary metabolismdefense; induced defense or acquired defense. Then differential gene expression is alsoimpacted by elicitors; now gene expression that responds to signals or triggers consequentlyforming enzymes concerned in the synthesis of pathway.Because now this is one of the ways which I said calcium metabolism intern can be relatedto transcription or translation; transcription of certain genes where which are responsiblefor reducing those enzymes or expression of those enzymes which may be directly involvedin the biosynthetic pathway or even in the production of PR proteins. Now PR proteinsin general are part of your defense. Now, mechanism of action again removal ofregulatory repressors; so in turn this may also impact removal of repressors which maynot be ; sometimes it is you will observe that even I told you about cyclo tides itis observed that under in vitro conditions if you elicit; add a stresssignal, then youmay end up getting compounds which are not known to be produced in the natural plant.Why is it happening? That is the very indication that these elicitors or your environmentalepigenetic factors may be responsible for in dueto remove the repressors or to inducethe promoters, they might be acting as repressors and inducers in the secondary metabolic pathways;thereby leading to expression of certain genes which are responsible for production of novelmetabolites. So, these genes might be remaining cryptic;so it may elicit those cryptic genes. So, removal of regulatory repressors, geneticmanipulation of enzymes involved in biosynthetic pathway or as metabolic inducers for increasesecondary metabolism. These are the different ways in which the elicitors can play a role.Therefore, till date elicitor adhesion is one of the most promising strategies to enhancethe yield of these in vitro cultures. So, under in vitro conditions they providethe stimulus which forms the basis of exploiting the biotechnological potential of these plantcells. Now, as I has; I have already mentioned binding indirectly now these elicitor moleculesthey may bind to the receptors indirectly inducing the transcription activity of thegenes involved in the calcium cyclic AMP signaling pathway.