Isoprenoids are a diverse group of compounds that have extensive applications in pharmaceuticals, flavours and flagrances. They are normally produced in organisms as primary or secondary metabolites. The current isoprenoid production methods include organic synthesis, extraction from plants and biosynthesis. Organic synthesis of isoprenoids is complex and expensive and extraction of isoprenoids from plants in limited by the small quantities which plants produce, high cost of purification and the geographical location of the desired plants. Biosynthesis using genetically modified organisms shows the promise of being a more economical and environmentally friendly way to produce isoprenoids. The pathway in Saccharomyces Cerevisiae to be modified is the pyruvate dehydrogenate bypass. In this path, pyruvate from glycolysis is converted into Acetyl CoA by the action of the enzymes pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and acetyl CoA synthase (ACS).Knocking down genes like PYC1, PDA1 and ADH1 which code for enzymes that divert the carbon flux from production of Acetyl CoA have been shown to have adverse effects on the cell such as poor growth. Pyruvate decarboxylase is also always abundant in the cell. Therefore, only the genes ALD6 for acetaldehyde dehydragenase, and ACS1 for Acetyl CoA will be overexpressed while no genes will be knocked down.Overexpression of ACS1 alone leads to a 8-23% increase in acetate production while overexpression of ALD6 alone leads to a 19times more ALD6 activity but a decrease in acetate(talk of Acetyl CoA instead) production. The coexpression of both genes gave just a 10% increase in Acetyl CoA production. However, the coexpression of ALD6 and the Salmonella Enterica variant of ACS1 gave an 80% increase in Acetyl CoA production. Overexpression of genes is done by the use of multi-copy plasmids. Genes are multiplied using polymerase chain reaction and cloned into sites on the plasmids. The transcription of ALD6 and ACS can be monitored by using cDNA or oligonucleotide arrays although cDNA is preferred for the higher binding specificity it offers due to the large size of its probes. Two dimensional gel electrophoresis can be used to determine the concentration of ALD6 and ACS1 produced while gas chromatography and mass spectrometry can be used to determine the levels of metabolites produced. For the metabolic flux analysis, mass balance at steady state is used to generate data for creating models which can be solved by computer algorithms. Finally, high performance liquid chromatography combined with mass spectroscopy can be used to determine the concentration of isoprenoids produced. This approach holds a lot of economic promise however, the long term effects of such genetic manipulation are yet to be assessed.
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Isoprenoids (also called terpenoids) are a class of organic compounds made up of two or more structural units derived from isoprene (Britannica 2009a). They are a very diverse group of compounds with over 40,000 identified (Withers and Keasling 2007). They are produced in all living things and represent the largest secondary metabolites that have been isolated from organisms (Ajikumar et al., 2008). Their function in organisms include electron transport, protein phenylation and in overall cellular and organism development (McCaskill and Croteau, 1997, 1998). An example of isoprenoids in animals is cholesterol while examples in plants are artemisinin, taxol and carotene.
Why Isoprenoids Are Compounds Of Interest
Because of the structural diversity of the isoprenoids, they have varied biological and chemical uses. Some areas where they have found extensive application include the following:
- Pharmaceuticals: Many novel drugs are either wholly or partly isoprenoids. Examples include Taxol, artemisinin, espintanol, 1, 8-cineole, linalool and menthol. Taxol has been used for chemotherapy for cancer patients, artemisinin is well known for its anti-malarial properties (Ajikumar et al., 2008).
- Food industry: Isoprenoids are used in the food industry mainly as colourants and flavourings. Examples include menthol, citral, carotenoids and squalene. Carotenoids are used as food colourants and anti-oxidants while β-carotene is a precursor of vitamin A in margarine, dairy products and soft drinks (Chemler et al 2006). Menthol is also widely used in sweets, chewing gums and other foods. Vitamins D and K are also made from isoprenoids (Berstenhorst et al).
- Fragrances: The isoprenoid citral is well known for its lemon scent which is used in detergents and cosmetic products. Others include geranoil (used in rose fragrances), limonene and camphor (Britannica 2009b).
- Feed supplements: Menadione, thymol, carvacrol, astaxanthin and carotenoids among others are used as supplements in animal feeds while Carvacrol is known to improve feed conversion in female broilers (Lee et al 2003).
- Varnishes and insecticides: Pyrethrins and prenylbisabolane diterpene among others are used for insecticide production (NPIC 1998).
Current Production Methods
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At the moment, isoprenoids are produced by three main methods and these are:
- Chemical synthesis: many isoprenoids are produced from different organic compounds, depending on the desired product. This production method has various disadvantages. Firstly, because of the complex nature of isoprenoids, very complex processes have to be designed for their production leading to high manufacturing costs (Shiba et al., 2007). Also, such processes typically have adverse effects on the environment in respect to waste such as process effluents and used catalysts (Chemler et al., 2006).
- Extraction from plants: isoprenoids have for a long time been extracted from plants. This process is geographically limited and plants naturally produce only small amounts of isoprenoids which makes purification and recovery costs very high (Kerby and Keasling 2008).
- Biosynthesis: In this more recent method, isoprenoids are produced by micro-organisms such as Escherichia Coli and Saccharomyces Cerevisiae, from inexpensive carbon sources like glucose (Wang et al., 1998). This method presents several advantages. According to Chemler et al (2006), since micro-organisms grow at a quicker rate than plants, greater amounts can be produced in less time. Also, organisms mainly used have little effect on the environment. Therefore, this method is both cheaper and more environmentally friendly and gives higher yields especially if genetically engineered organisms are used (Chemler et al., 2006).
System Biology Optimization Strategy
The main focus is to maximize the production of Acetyl CoA, a precursor in the production of isoprenoids, using Saccharomyces Cerevisiae. Therefore, the pyruvate dehydrogenase bypass will be optimised. The Acetyl CoA produced enters the mevalonate (MV) pathway where it is converted in a series of steps to isoprenoids (Shiba et al., 2007). The diagram below shows the pyruvate dehydrogenase bypass marked by the bold arrows.
Glucose is broken down in the cytosol via the Embden-Meyerhof-Parnas pathway to give pyruvate. Part of this pyruvate enters the mitochondrion where they enter the citric acid cycle and is used for energy production by the cell.
In the pyruvate dehydrogenase bypass, the first reaction (conversion of pyruvate to acetaldehyde) is catalysed by the enzyme pyruvate decarboxylase. According to Withers (2007), this enzyme is usually in abundant amounts even in glucose-limited conditions because the oxidation of glucose to ethanol is very favoured in Saccharomyces Cerevisiae Therefore, our focus will be on the enzymes catalysing the other reactions. The next point is the branch point where acetaldehyde is converted to ethanol and acetate, both reactions catalysed by different enzymes. The conversion of acetaldehyde to acetate is catalysed by the enzyme acetaldehyde dehydrogenase. The acetate formed is then converted to Acetyl CoA in a reaction catalysed by Acetyl CoA synthase. For more isoprenoid formation, the carbon flux has to be shifted from the ethanol synthesis reaction to the Acetyl CoA synthesis reaction and this will be done by the overexpression of acetaldehyde dehydrogenase and acetyl CoA synthase (Shiba et al., 2007).
Analysis And Modelling Of The Metabolic Pathways
The metabolic pathway for the production of Acetyl CoA is given below.
Pyruvate from the glycolytic pathway is converted to either oxaloacetic acid in the mitochondria, Acetyl CoA in the mitochondria or acetaldehyde in the cytosol.
The conversion of pyruvate to oxaloacetic acid is catalysed by the enzyme pyruvate carboxylase (PYC) which has by two isozymes, PYC1 and PYC2. The oxaloacetic acid produced from the reaction enters the citric acid cycle and is used for cellular respiration. Therefore, knocking down the genes coding PYC1 and/or PYC2 will result in reduce the ability of the cell to respire. This will probably rob the cell of the energy it needs to function and thereby lead to poor cell growth.
Likewise, knocking down the genes that code for the enzymes PDA1 and PDB1 which catalyze the conversion of pyruvate to mitochondrial Acetyl CoA will have very little change in the net production of Acetyl CoA. According to Hohmann (1991), although the knockdown of the PDA1 will reduce direct conversion of pyruvate into mitochondrial Acetyl CoA, the Acetyl CoA formed in the cytosol from the pyruvate dehydrogenase bypass will be transported into the mitochondria for use in the Krebs cycle. Therefore, there will not be a significant overall increase in the amount of Acetyl CoA available for isoprenoid production.
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For the conversion of acetaldehyde to ethanol, the enzyme that catalyses the reaction is alcohol dehydrogenase, which has seven isozymes. According to Shiba et al (2007), ADH1 is the main isozyme that is involved in the forward reaction while ADH2 catalyzes the reverse reaction (conversion of ethanol to acetaldehyde). The other isozymes function in varying capacities and when certain physiological conditions are prevalent (Shiba et al., 2007).The downregulation of ADH1 will lead to lower ethanol production and shift the carbon flux in favour of Acetyl CoA production. However, Hohmann (1991) reported that knocking down the gene coding for ADH1 gave between 20- 40% lower ethanol production than wild type strain but cells also showed impaired growth.
Consideration Of Genes For Upregulation
1. PDC1, PDC5 and PDC6: These enzymes code for the enzyme pyruvate decarboxylase which catalyzes the conversion of pyruvate to acetaldehyde. According to Pronk et al (1996), pyruvate decarboxylase is always abundant in the cell even in glucose-limited conditions. Therefore, we can leave out upregulating the pyruvate decarboxylase genes as it is not the limiting step.
2. ALD6: This is the gene that codes for acetaldehyde dehydrogenase which catalyzes the conversion of acetaldehyde to acetate. This gene will be upregulated to divert the carbon flux from the ethanol-producing reaction to the acetate-producing reaction. According to Shiba et al, mutant containing multicopy ALD6 plasmid shows 19 times more ALD6 activity but caused a reduction in cell mass (Shiba et al., 2007). Therefore upregulating ALD6 effectively increases the carbon flux in the acetate production direction.
3. ACS1: This gene codes for Acetyl CoA synthase. According to Shiba et al, the other isozyme of enzyme ACS2 has a higher Michaelis -Menten constant (lower affinity for acetate) and lower Vmax. Therefore, ACS1 alone can be overexpressed to give the increased production wanted. Shiba et al (2007) found that when ACS1 alone was overexpressed, the isoprenoid production increased by between 8-23% and cell growth was unaffected.
Overexpression Of Ald6 And Acs1
Shiba et al (2007) found that when ALD6 and ACS1 were coexpressed, there was only a 10% increase in the amount of isoprenoid (amorphadiene in this case) produced. It was suggested that ACS1 was post-translationally regulated. However, when the Salmonella Enterica variant of ACS1 was coexpressed, there was as increase in isoprenoid production of 80% (Shiba et al., 2007).
Limitations Of Using A Systems Biology Approach
1. It is difficult to assess the long term impact of altering gene expression in a cell. Organisms including Saccharomyces Cerevisiae have evolved over billions of years, developing the pathways available in them for a more efficient existence. Therefore, altering those genes and pathways could have adverse implications on the cell which even though not visible in the short term might become more evident in the long term ().
2. Because of the number and complexity of the metabolic pathways in the cell, deleting a gene or upregulating another could affect a metabolite downstream of the alteration point, which is vital for the proper functioning of the cell ().
3. The behaviour of Saccharomyces Cerevisiae depends on its environment not just its genetic expression. Therefore altering just the genetic make-up without providing the cell with an optimal environment might not give the best isoprenoid production by the cell ().
4. Altering the gene expression of an organism can make it vulnerable to conditions it was originally unaffected by (). Therefore, the presence of certain chemical substances (which it was originally resistant to) in its environment might harm the cell.
How To Alter The Levels Of Gene Expression
To overexpress the genes ALD6 and ACS1, multicopy plasmids are used. The gene is isolated from Saccharomyces Cerevisiae and multiplied by polymerase chain reaction(PCR) using a primer ALD6-1 (in the case of ALD6.find that for ACS1 ).The host plasmids are treated with a restriction enzyme and the genes(ALD6 and ACS1) are cloned into appropriate sites(BamHI and Smal for ALD6) (Shiba et al., 2007).
Monitoring Of Transcription Of Ald6 And Acs1
There are two methods by which gene transcription can be monitored. They are
1. Use of cDNA arrays
2. Use of Oligonucleotide arrays
cDNA arrays are prepared by placing DNA strands (hundreds to thousands) on a glass or nylon micro plate (Cheung et al., 1999, as cited by Paszek 2007a ). Cells are centrifuged to remove growth medium. mRNA can then be extracted using a mRNA purification kit (Ambion 2009) and modified with fluorescent dyes to form probes which are then hybridized with cDNA (Paszek 2007a). Chip is then analyzed using coloured lasers to find out which genes were transcribed (Paszek 2007a). Probes used in this technique are longer than those used in oligonucleotide arrays ().
The other method which can be employed is the use of oligonucleotide arrays. These arrays use small oligonucleotides which can detect very small amounts of RNA and this process is readily scalable to monitor simultaneously tens of thousands of genes (Paszek 2007b).
Quantifying Enzyme Production
THE quantification of enzymes can be done by either gel electrophoresis, mass spectrometry or a combination of both methods or x-ray crystallography (). Gel electrophoresis can be done in either one or two dimensions but two-dimensional gel electrophoresis is preferred. Seperation of the enzymes is done first by isoelectric point (as different enzymes have different isoelectric points) while the second separation is done by molecular weight (Grover 2009). Mass spectrometry gives the amounts of the different enzymes using differences in molecular weight ().
Determining The Levels Of Metabolites And Flux Through The Central Carbon Metabolism
The amounts of metabolites produced can be determined by gas chromatography and mass spectrometry. Gas chromatography uses an inert gas as the mobile phase to separate the complex mixture into its components on a stationary phase, typically a solid which has a high boiling point liquid absorbed on it (Clark 2007). Mass spectrometry can then be used to determine the different metabolite concentrations of identified metabolites. The changes in carbon flux along the metabolic pathway can be determined by comparing the amounts of the different metabolites produced from wild-type strain and the different mutants of Saccharomyces Cerevisiae. To carry out a metabolic flux analysis, steady state condition in the cell is assumed, a mass balance is carried out and data obtained is use to create models that describe the flux distribution (Grover 2009). Computer programmes and algorithms like linear programming can then be used for the analysis.
Determination Of Final Product Concentrations
A combination of high performance liquid chromatography and mass spectrometry (HPLC-MS) will be used to determine the product (isoprenoid) concentration. High performance liquid chromatography (HPLC) is an upgraded form of column chromatography in which the solvent is forced down the column by pressures up to 400 atmospheres rather than having it drip through, making the process faster (Clark 2007). It is more advantageous for use in quantifying isoprenoid concentrations as it does not require special sample preparation unlike gas chromatography which requires conversion of the sample mixture to a stable vapour (Lindsay 1997). there are two types of HPLC namely: normal HPLC, which use a polar stationary phase(typically fine silica), and reverse HPLC in which non-polar molecules are attached to the stationary phase to increase its interaction with non-polar components of the mixture to be separated(Clark 2007).Reverse HPLC is the appropriate method for quantifying isoprenoid concentration since isoprenoids are non-polar. The sample is introduced at the top of the column, eluted with an appropriate solvent. The different components of the mixture are detected by a UV detector based on their UV absorption. The identified components (including the isoprenoids) are then sent to a mass spectrometer which identifies and quantifies the various products (Lindsay 1997). A combination of gas chromatography and mass spectrometry may also be used instead of HPLC-MS.
The pathway for maximising Acetyl CoA production in Saccharomyces Cerevisiae is the pyruvate dehydrogenase bypass which converts pyruvate from glycolysis to Acetyl CoA. The enzymes ALD6 and the Salmonella Enterica variant of ACS1 were overexpressed and gave the 80% more Acetyl CoA than the wild type strain (specify) (Shiba et al., 2007). However, the long term effects of using a system biology approach are yet to be assessed.
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