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Malaria is a mosquito-borne infectious disease that is caused by a parasite known as the Plasmodium falciparum. The parasite enters the human body via saliva of an infected female mosquito.
Malaria is considered to be a global health crisis as it threatens 300-500 million people and causes more than one million deaths every year [Who,2005]. It can either be controlled and cured with naturally occurring therapeutics or synthetically made antimalarial drugs that are currently being developed whose efficacy against malaria is yet to be confirmed by stringent clinical Testing (Vennerstrom, J. L. et al, 2004)
It was required to develop a yeast-based bioprocess that is able to produce Artemisinin for antimalarial treatment. The bioprocess is required to use glucose as the feedstock and yeast cells as the client already has subsidiaries working with yeast, as such it is considered to be cost effective.
However from bioprocessing point of view, Saccharomyces cerevisiae is qualified as the expression system because:
S. cerevesie is characterised by intracellular compartments that ensure that the mechanism required for either secretion or insertion of the host protein in the plasma membrane.
Both A.annua and S. cerevesie utilises the Mevalonate Pathway (MVA) to generate IPP and DMAPP. (discussed further below)
But more importantly, S. cerevisiae is favoured over prokaryote because organisms such as E.coli produces toxic and contains pyrogenic cell wall components which effectively undesirable in the production of pharmaceutical or food components (Scheiner-Bobis, 2009)
Artemisinin is a naturally found therapeutic medicine that is highly effective towards multi-drug resistant Plasmodium falciparum. It is extracted from Artemisia annua, commonly known as the sweet wormwood. Chemically, artemisinin is a sesquiterpene lactone containing an unusual peroxide bridge, which is believed to be responsible for the drug's mechanism of action. Only few other natural compounds with such a peroxide bridge are known. (RSC, 2006)
Why Bio synthesis?
Naturally the concentration of artemisinin in A. annua, ranges from 0.01% to 0.8% of the plant dry weight, making artemisinin relatively expensive and difficult to meet the demand of over 100 million courses of artemisinin-based combinational therapies per year. One way to overcome this problem is to synthesise artemisinin chemically using basic organic reagents which is however complex and commercially not viable. (Schmid., Hofhienz., 1983)
Alternatively, biosynthesis of artemisinin or any of its derivatives, from microbially sourced artemisinic acid (its immediate precursor) have shown to be cost-effective, high-quality and reliable source of artemisinin. (Acton., Roth., 1992)
The Metabolic pathways
For biosynthesis, the metabolic pathways of A.annua, require to be understood so that the key enzymes and the intermediates responsible in production of artemisinin can be identified.
Isoprenoids, also known as terpenoids are a large and diverse class of naturally occurring organic chemicals. Each member of this class is assembled from 5-carbon (C5) isoprene units and derived metabolically from the basic building block, isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). These two building blocks serve as the basis for the biosynthesis of molecules used in vivo in diverse processes.
Isoprenoids also form the largest group of so-called secondary metabolites, such as the extremely diverse classes of plant defensive terpenoids that are widely exploited as perfumes, food additives, and pharmaceutical agents such as the antimalarial compound artemisinin. (Gershenzon, 2007)
The basic two isoprenoid building blocks, IPP and DMAPP are generated in cells by one of two distinct biosynthetic routes. The mevalonate pathway (MVA) and the non-mevalonate pathway (MEP). Both have distinct evolutionary origins and are phylogenetically compartmentalised. (Boucher., Doolittle., 2000)
Archaebacteria and most eukaryotes including all metazoans and fungi, use the MVA pathway. While the vast majority of eubacteria use the MEP pathway, including the key pathogens. Plants on the other hand, synthesise IPP and DMAPP using both pathways. (Audrey R., 2011)
For biosynthesis engineering, the MVP pathway was chosen as it is present in both A.annua and the Yeast.
In mevalonate pathway, Acetyl-CoA undergoes condensation with another acetyl-CoA subunit via Acetyl-CoA Transferase to form acetoacetyl-CoA. Acetyl-CoA condenses with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by HMG-CoA synthase. HMG-CoA is then reduced to mevalonate using HMG-CoA reductase by the help of NADPH. This is important because HMG-CoA reductase is the rate limiting enzyme of the MVP pathway. Mevalonate kinase catalyse the reaction of Mevalonate to 5-phosphomevalonate which is then converted to 5-pyrophosphomevalonate by phosphomevalonate kinase. (Audrey R., 2011)
Isopentenyl pyrophosphate (IPP) is formed from 5-pyrophosphomevalonate, which is catalysed by an enzyme called 5-pyrophophomevalonate decarboxylase.
IPP is then converted to its highly electrophilic isomer, dimethylallyl diphosphate (DMAPP), by isopentenyl pyrophosphate isomerase. DMAPP is a crossover product between both the two pathways and is then catalysed by Farnesyl diphosphate synthase into Geranyl pyrophosphate (GPP) which is substrates for the successive reaction that results in the synthesis of farnesyl diphosphate (FPP). (Audrey R., 2011)
Biosynthesis of artemisinin involves using engineered Saccharomyces cerevisiae to produce high concentration of (preferably more than 100 mg.l-1) of artemisinic acid. It achieved by converting FPP to amorphadiene which subsequently oxidises into artemisinic acid by a cytochrome.
To ensure the genetic stability of the host strain; all of these modifications were made by chromosomal integration of the host yeast stain and was performed in three steps;
Engineering the farnesyl pyrophosphate (FPP) biosynthetic pathway (MVA) to be overexpressed in order to increase FPP production while decreasing its use for sterols.
Introducing the amorphadiene synthase gene (ADS) from A.annua into the high FPP producer to convert FPP to amorphadiene.
Cloning a cytochrome P450 that performs a three-step oxidation of amorphadiene to artemisinic acid from A.annua and expressing it in the amorphadiene producer. (Ro et al., 2006)
Yeast engineered with ADS alone is reported by Ro et al., 2006 to produce a low quantity of amorphadiene (4.4 mg.l-1). However, to increase FPP production, the expression of several genes responsible for FPP synthesis was up-regulated and instead of complete removal, the gene responsible for FPP conversion sterols was down-regulated. Sterol leads to ergosterol production which is important for plasma membrane structure, function and for localisation of plasma membrane proteins in Yeast cells.
According to Iwaki et al., 2008 by downregulating the conversion of FPP conversion; only small amount of ergosterol will be produced, just enough for the cell to sustain. The remaining FPP can then be converted to more amorphadiene which subsequently oxidises to artemisinic acid.
To create a strain that could produce artemisinic acid from amorphadiene; genes encoding for enzymes responsible for oxidising amorphadiene to artemisinic acid in A. annua were had to be identified.
Engineering modification of MVP pathway of S.cerevisaie can be performed by overexpressing the soluble form of 3-hydroxy-3-methyl-glutaryl-coA reductase enzyme (HMGR) which is the rate determining enzyme and responsible for FPP conversion to sterol. According to Ro et al, 2006 overexpressing improves amorphadiene production approximately by fivefolds. Such modifications, even though improve the amorphadiene production, it however cause decrease in cell density due to insufficient production of ergosterol for Yeast cells to sustain. As such, integration of an additional copy of HMGR into the chromosome can be achieved which increases amorphadiene production drastically yet preventing decline in cell density. As a result, the combined modifications result in significantly high amount of amorphadiene and a sesquiterpene (SQS) production nearly 500-fold higher than the yeast engineered with ADS alone. (Jackson et al., 2003)
Similarly, ERG9 gene which encodes squalene synthase (also known as FPP farnesyl transferase) can be downregulated using methionine-repressible promoter (PMET3) allowing the sterol production to be reduced while increasing the FPP conversion to amorphadiene by twofolds. (Ro et al., 2006)
Also, a semi-dominant mutant allele (upc2-1) that enhances the activity of UPC2 can be overexpressed. According to Davies et al., (2005), UPC2 is a global transcription factor regulating the biosynthesis of sterols in S. cerevisiae and has a mild effect on amorphadiene production.
Studies have shown that the combination of downregulating ERG9 and overexpressing of upc2-1 further increase amorphadiene production by significant amount (further 80%). (Ro et al., 2006)
Monooxygenase (P450) of cytochrome P450 group, catalyses the first stereospecific hydroxylation of amorphadiene in A. annua. (Bertea et al., 2005)
P450 is a conserved Asteraceae sesquiterpene lactone biosynthetic enzyme. Its corresponding full-length cDNA (CYP71AV1) can be recovered from A. annua DNA. The cDNA encodes an open reading frame of 495 amino acids.
Note: According to Phylogenetic analysis; CYP71AV1 shares a close ancestry with other P450s that catalyse the hydroxylation of monoterpenoids (CYP71D13/18; ref. 19), sesquiterpenoids (CYP71D20; ref. 20) or diterpenoids (CYP71D16; ref. 21). This further suggests the possible involvement of this P450 gene in terpenoid metabolism.
In order to have a functional heterologous expression of CYP71AV1; Cytochrome P450 oxidoreductase (CPR) must also be isolated from A. annua.
Studies have shown and confirm that structurally authentic artemisinic acid is synthesised by transgenic Yeast de novo, using A. annua CPR as a redox partner for CYP71AV1. Similarly, the three-step oxidations by P450 enzymes have been previously reported in plant hormone gibberellin biosynthetic pathways. (Helliwell et al., 1999: Helliwell et al., 2001)
A similar approach has been reported previously by Eelco Wallaart et al, 2000. Where cDNA clone encoding amorpha-4, 11-diene synthase was isolated from A.annua and expressed in E.coli. The recombinant enzyme demonstrated the conversion of amorpha-4,11-diene to farnesyl diphosphate.
Likewise, experiments have been conducted in vitro to check whether CYP71AV1 catalyses all three oxidation reactions from amorphadiene to artemisinic acid. Ro et al., 2006 reported such observation when microsomes from engineered S. cerevisiae strain expressing CPR and CYP71AV1, were incubated along with pathway intermediates (amorphadiene, artemisinic alcohol and artemisinic aldehyde).
The attempt illustrated efficient conversion of amorphadiene, artemisinic alcohol and artemisinic aldehyde to the final product artemisinic acid in microsomes containing CYP71AV1 and CPR. This demonstrates explicitly the ability of recombinant CYP71AV1 to catalyse three oxidation reactions at the C12 position of amorphadiene. (Bertia et al., 2005)
Similarly, efficient conversion of amorpha-4, 11-diene to artemisinic acid in vivo by recombinant CYP71AV1 indicates that the membrane-bound, multifunctional CYP71AV1 is a key contributor to artemisinin biosynthesis.
HmgR Phosphorylation inhibition
Short term down-regulation is achieved by inhibition of HMGR by inhibition by phosphorylation at Serine residue. AMP- activatd protein kinase phosphorylate HMG-CoA reductase at Serine 872 residue, inactivating the enzyme. Phosphorylation occurs when energy charge is low in the cell there is a rise in AMP concentration. However, HMGR is the rate determining step and is responsible for FPP conversion to sterol which subsequently converted to artemisinic acid. HMGR Phosphorylation would have detrimental effect in production rate of artemisinic acid.
To inhibit phosphorylation; site-specific mutagenesis can be performed to DNA sequence of HMGR gene. Serine residue, R - (CH2) -OH can be replaced with Alanine residue,
R- (CH2)-H. Serine 872 (AGA) into alanine (CGA).
Assuming the modification will not change protein shape nor its active site, Phosphorylation cannot occur and structural conformation will not occur. As such, up-regulation of the artemisinic acid formation pathway can be achieved.
Modified genes including the heterologous genes taken from A.annua, can be inserted into the S.cervisiae using Molecular cloning.
Molecular cloning involves cloning of any DNA fragment into a host organism and it essentially involves several steps.
A cloning vector such as plasmid is used to as a vehicle to artificially carry the genetic material into the host organism. In the case of S.cerevisiae an expression plasmid should be used as it contains an origin of replication which assures the transcription and translation events to occur. (Brown., 2006)
Nevertheless, almost always; vectors contain four DNA segments that are critically important to its function.
an origin of DNA replication including the recombinant sequences, to replicate inside the host organism
One or more unique restriction endonuclease recognition sites that serves as sites where foreign DNA may be introduced
A selectable genetic marker gene that can be used to enable the survival of cells that have taken up vector sequences
An additional gene that can be used for screening which cells contain foreign DNA.
The expression plasmid is treated with a restriction endonuclease to cleave the DNA at the site where the engineered sequence can be inserted. The restriction enzyme is chosen to generate a configuration at the cleavage site that is compatible with that at the ends of the rDNA. This is done by cleaving the expression plasmid DNA and rDNA with the same restriction enzyme, for instance EcoRI. (Russell., Sambrook., 2001)
The genomic DNA to be cloned is extracted from the A.annua and then purified using simple methods to remove contaminating proteins. Polymerase chain reaction (PCR) methods are often used for amplification of specific DNA or RNA (RT-PCR) sequences prior to molecular cloning.
The purified genomic DNA along with the modified mevalonate pathway sequence is then treated with a restriction enzyme to generate fragments with ends capable of being linked to those of the expression plasmid.
Once completed, the transformation of S.cerevisiae can be carried out using electroporation. Electroporation involves using high voltage electrical pulses to translocate DNA effectively (reportedly: Delorme, 1989) across the cell membrane/cell wall.
Even so, only a small fraction of the cells will actually take up DNA and as a result; artificial genetic selection is used where cells that can actively replicate encoded DNA can be isolated using selectable marker gene.
During bio-processing, the engineered S.cerevisiae cells are required to be isolated from the product upon process termination. This can be done either by (1) suspending cells in a solution and compartmentalised by a membrane in a reaction vessel (2) immobilising within or on the membrane matrix itself, where the membrane acts as a support for cells and a separation unit.
Cell immobilisation is a well known technique used in many industrial applications. Immobilisation can be done in or on the membrane by entrapment, gelification, physical adsorption, ionic binding, covalent binding or cross-linking. (Giorno., Drioli., 2000)
In pharmaceutical industry, immobilisation usually carried out by entrapment. It is used in bioreactors for the entrapment of plant protoplasts, bacteria, enzymes, drugs etc. One of the examples is the production of ampicillin and amoxicillin by entrapping penicillin Amidase in cellulose triacetate fibres by ionic network formation. This further demonstrates the versatile practicality of entrapment technique and as such it should be implemented for the immobilisation of S.cerevisiae.
Entrapment by ionic network formation
Ionic network formation involves single-step entrapment where gel beads are formed using polysaccharide. Alginate is a linear polysaccharide that forms a stable gel in the presence of positively charged ions such as calcium cations.
Gel beads are formed by thoroughly suspending yeast cells in a sodium alginate solution. Yeast-alginate solution is then dripped into a stirred crosslinking calcium chloride solution. Gel bead formation occurs as the yeast-alginate drops come in direct contact with the calcium chloride solution. Once formed, the beads are air dried followed by wash with sterile saline solution to remove excess calcium ions and cells. (Wang., 2009)
The small diameter beads are generally preferred because of the favourable mass transfer characteristics. The Smaller the beads the higher the surface area to volume ratio, allowing good transport of essential nutrients and are also less fragile upon mild agitation. Diffusion limitations within larger beads may limit cellular metabolism as the lack of essential substances like oxygen supply to the interior of the beads may lead to cell death as a result of consumption from the surrounding cells. Therefore a good control of bead size and shape is crucial and should be carefully controlled. A suitable methodology for production of small porous beads under controlled conditions is also necessary. Studies conducted by Zain, et al, (2011) on immobilised yeast cells by calcium alginate in the production of ethanol, shows that the highest theoretical yield was obtained using alginate beads with a diameter of 0.5 cm with the volume of beads to the volume of alginate media ratio of 0.4 and concentration of 150 g/L of alginate solution.
Single-step method is quite simple to achieve, however the preparation usually suffer from heat damage and low mechanical strength. Alternatively, two-step methods may be employed. The procedure involves the use of resin and curing agent to create a more porous structure beads with desirable elastic behaviour and improved mechanical properties throughout handling. (Klein., Eng., 1979)
Advantages and Disadvantages
Free suspended yeast cells are expected to be disrupted by mechanical agitation or liquid shear forces in the vessel. This shear sensitivity means that mechanical agitation may be detrimental to cells and that feedstock cannot be transported using conventional pumps without significant loss of viability. Immobilisation provides solution to such problems as it protects yeast cells from sustained shear with no equipment modification. (Williams., Mavituna., 1992)
Similarly, the physical separation of the cells and medium in the immobilised system also makes for easy exchange of medium for purposes of metabolic control or nutrient replenishment. The composition of the culture medium can be readily and continuously monitored via an external loop, and the concentrations of O2 or sugar can be adjusted as required. Similarly, extracellular products can be collected continuously (in continuous process) by adsorption on a suitable resin, or by other means (Tyler et al., 1995)
From system stability point of view, the entrapment immobilisation provides a stable colloidal system with beads being suspended in the medium. Assuming that oxygen, glucose and artemisinic acid have no polar interaction contribution; the beads will not flocculate due to their relatively large sizes but more importantly due to cross linking of Ca cations forming a positively charged surface, with suppressing Van der Waals attractive forces interaction between beads. As a result repulsive forces dominate and keep the beads suspended, as shown ---- below. The suspended system is said to have zeta potential far away from the iso-electric point (iep) where particles carry no net electrical charge.
Other advantages of immobilisation include increased reactor stability and productivity, improved product purity and quality, and reduction in waste.
The main disadvantage of immobilisation is that high productivity is achieved only by the cells suspended near the surface of the beads. They excrete product of interest which diffuse into the culture medium. Attempt to induce the released product from cells suspended in the centre, is normally techniques such as permeabilisation which decrease cells' viability to an undesirable extend. (Hutabarat, 2001)
S.cervisiae reproduce asexually about every 90 minutes which effectively means that the process must be terminated after that period otherwise the increase in cell density can lead to mechanical disruption of the immoblised matrix.
However, in a continuously operated immobilised system; the disengagement of growth and production phases means that productive cells cannot compete with the nonproductive cells fermenter because instead of reproducing themselves to propagate further; the productive cells spend the nutritional and energy resources producing chemicals in quantities far above the amount necessary for their survival, allowing the cells in the bioreactor to be retained for extended periods. (Williams., Mavituna., 1992: Wang., 2009)
Aeration of Immobilised Cells
Oxygen is a key nutrient in cellular metabolism. While low oxygen concentration affects cell growth and overall product yield, oxygen in excessive amount can potentially be toxic to the cells. (Sadettin, 2005)
Glacken et al. attributed the toxicity to the generation of superoxide radicals that may affect cells directly or indirectly by oxidizing medium components. The oxygen requirement of living cells varies according to different cell lines and culture conditions.
The oxygen demands of plant and microbial cell cultures are several orders of magnitude higher than animal cell cultures, making oxygenation one of the primary challenges in cell culture scale-up. The importance of aeration increases with the escalation of bioreactor size and cell concentration.
The classical and typically used aeration method is sparging, which is bubbling gas directly into the culture medium for liquid volumes >10-100 L. Other methods are usually problematic, although they are able to outperform sparging in some aspects. (Sadettin, 2005)
Alginate beads consist of hydrophilic polymer chains cross linked with cross-linking agent (Ca cations). The degree of water sorption of a hydrogel is not only determined by chemical composition but also by the physical forces and subsequent elastic responses of the constituent macromolecular chains. Studies conducted by ---- have shown that a variation in chemical composition of the network can effectively control the swelling of the hydrophilic macromolecular matrices (references)
According to ---- the swelling ratio constantly increases up to 33.3% of alginate content of the bead while beyond this amount a fall in swelling ratio is noticed. The decrease observed beyond 33.3% of alginate content may be attributed to the fact that much higher amount of alginate in the matrix gives rise to a compact network of bio polymeric chains which because of greater interaction among the alginate molecules results in a restrained mobility of the network chains. However, a compact structure give rise to small pore sizes of the network that also slows down diffusion of molecules into or out of the bead as such it brings a fall in the swelling ratio.
Also, the degree of water sorption constantly decreases with increasing concentration of cross-linker. The observed fall in the swelling ratio may be attributed to the fact that with increasing number of calcium ions in the cross-linking bath, the alginate beads containing smaller cavities are produced which accommodate less amount of water and thus decrease the swelling of the polymer beads.
Effect of pH and temperature
Alginate beads are said to be structurally stable around pH 4-4.5. Increase in pH affects their structural stability. Beyond pH 7.2, the higher alkaline range causes the COOH groups of alginate molecules to undergo dissociation which weakens the physical forces between the two biopolymer, which leads to disintegration of bead and possibly flocculation as the zeta potential is decreased and the system is likely to reach its iep.
Similarly, increase beyond the optimum temperature weakens the cross-links causing the bead to integrate. Attributed to the continuous erosion of surface layers of the swelling beads. It is worth mentioning the fact that the erosion of bead becomes faster with increasing temperature due to greater solubility at higher temperature due to greater solubility at higher temperature and thus erosion starts earlier at higher temperature.
The analysis of mass transfer in immobilised cell reactors is of great importance as it dictates the performance in these reactors which is often affected by;
The rate of transport of reactants to and products from the immobilised cell system due to external mass transfer limitations
the rate of transport inside the immobilised cell system due to internal mass transfer limitations
Internal mass transfer limitations are often more difficult to eliminate and their understanding is crucial for the analysis and optimisation of immobilised cell system performance.
Two physical phenomena are involved in the mass transport of molecules, namely diffusion and convection. Diffusion is the random motion of molecules that arise from thermal energy transferred by molecular collisions. Convection is a mechanism of transport resulting from the bulk motion of fluids.
Internal mass transfer
Internal mass transfer occurs by diffusion which is the random motion of molecules that arise from thermal energy transferred by molecular collisions. Diffusion occurs due to concentration gradient of a given substance per unit area. It can be described by Fick's first law, which states that the rate of transfer of the diffusing substance through a unit area is proportional to the concentration gradient measured normal to the section.
Where, J is the flux equals to the mass transfer rate per unit area of a section. C is the concentration of diffusing substance in x direction of the 3D Cartesian coordinate system. D is the diffusion Coefficient.
To conduct a mass balance on alginate beads, it was assumed that the beads are porous and spherical which allowed one to model the beads as catalyst pellets.
The internal mass transfer model for glucose, oxygen and artemisinic acid are almost similar except that both glucose and oxygen are diffusing in while artemisinic acid is diffusing out, giving rise to counter current flow of nutrients to products across the bead.
As such, internal mass transfer for nutrients and product gives;
Where DEff is the effective diffusion coefficient, dS is the change in concentration of substrate (glucose) along the bead's radius, r
It can be assumed that the reaction proceeds according to Michealis Menten kinetics, Ê‹, which is the rate of enzymatic reactions and is related to [S], the concentration of a substrate S and is given by:
Thus, is replaced by Ê‹ to give;
Where, Vmax represents the maximum rate achieved by the system, at maximum (saturating) substrate concentrations, Km is the Michaelis constant and represent the substrate concentration when the reaction rate is half of Vmax.
For the above derivation, the concentration of substrate at the surface of bead is taken as the film layer substrate concentration.
Complete derivation is provided at the appendix
External mass transfer
Mass transport of nutrients and the removal of products from the bead surface, is usually occured by convection. External mass transfer is described in terms of a liquid phase mass transfer coefficient ks, lumping convective, and diffusive mass transport in the boundary layer around the bead.
The average flux of substrate (Ns) to the fluid-solid interface may be written as:
kS - Average mass-transfer coefficient
S0 - Substrate concentrations in the bulk fluid
S* - Substrate concentration at solid-liquid interface.
At steady state, the reaction rate equals to the rate of glucose transported to the bead surface
The above derivation represents the transport of nutrients from bulk to the interface only. Complete derivation is given in appendix. It is worth mentioning the fact that in external mass transfer the liquid film interface were to be taken into account; as such the beads were considered as an uncharged, non-porous particle with the entire surface of which is uniformly accessible.
Simultaneous mass transfer
Both the internal and external mass transfer resistance are significant and occur simultaneously. To obtain a combined analysis which consider both resistance and provide a dynamic model for substrate mass transport; the boundary conditions at each step were change to dimensionless form.
Applying dimensionless boundary conditions
And combining equation 1 and 2, we get;
The Biot number, Bi, is representative of the relative degree to which the two resistances control mass transfer. Thiele modulus is the ratio of the intrinsic chemical reaction rate in the absence of mass transfer limitation to the rate of diffusion through the particle. Similarly, the physical interpretation of f2 is analogous to the Damkohler number which represents a ratio of maximum reaction rate to maximum flux through the diffusion layer.
The simultaneous mass transfer equation is valid for beads with thin-liquid film at the interface. For a fluid flowing past a solid, the whole resistance to mass transfer is in the stagnant liquid film such that that the actual mass transfer coefficient ks is related to the liquid film thickness δ as
In summary, we have created a strain of S. cerevisiae capable of
producing high levels of artemisinic acid by engineering the FPP
biosynthetic pathway to increase FPP production and by expressing
amorphadiene synthase, a novel cytochrome P450 and its redox
partner from A. annua.
The transgenic yeast produced artemisinic acid at a biomass fraction comparable to that produced by A. annua
(4.5% dry weight in yeast compared to 1.9% artemisinic acid and
0.16% artemisinin in A. annua) but over a much shorter time (4-5
days for yeast versus several months for A. annua). As such, the
specific productivity of the engineered yeast strain is nearly two
orders of magnitude greater than A. annua.
and its biochemical function was confirmed in vitro.
(Michaelis-Menten constants (Km) for cytochrome c and NADPH
were determined to be 4.3 ^ 0.7 mM and 23.0 ^ 4.4 mM (mean ^
s.d., n ¼ 3), respectively.)