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Biosynthetic production of artemisinic acid, an immediate precursor of antimalarial drug, known as Artemisinin is presented. The use of engineered yeast strain to produce artemisinic acid have shown to be cost-effective, high-quality and reliable source of artemisinin. FPP biosynthetic pathway of S.cerevisiae was modified, to overexpress and increase FPP production while decreasing its use for ergosterol. Amorph-4, 11-adiene synthase gene (ADS) from A.annua was introduced in host yeast strain to convert high FPP production into amorphadiene. Cytochrome (P450) plus a CPR were cloned to from A.annua to perform a 3-step oxidation of amorph-4, 11-adiene to artemisinic acid when expressed in the host yeast strain. Also, immobilisation of yeast cells by entrapment using sodium alginate and with calcium cations has made known to be beneficial in providing system stability, high purity & quality product and effortless downstream separation.
Table of Contents
It was required to develop a yeast-based bioprocess that is able to produce Artemisinin for antimalarial treatment. It was required to use yeast cells with glucose as feedstock as the client has subsidiaries already working with yeast, as such it is considered to be cost effective. However, from bioprocessing point of view, use of Saccharomyces cerevisiae is qualified as the expression system because;
S. cerevesie is characterised by intracellular compartments that ensure the mechanisms required for either secretion or insertion of the host protein in the plasma membrane.
Both A.annua and S. cerevesie use the Mevalonate metabolic Pathway (MVA) to generate IPP and DMAPP. (discussed further below)
S. cerevisiae is favoured over prokaryote because organisms such as E.coli produces toxic and contains pyrogenic cell wall components which makes them undesirable in the production of pharmaceuticals 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. Artemisinin is a sesquiterpene lactone and contains an unusual peroxide bridge, which is considered to be responsible for the drug's therapeutic effect. Only few other natural compounds are known with such a peroxide bridge. (RSC, 2006)
Why Bio synthesis?
Naturally, 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 (immediate precursor, artemisinic acid), using microbial cells have shown to be cost-effective, high-quality and reliable source of artemisinin. (Acton., Roth., 1992)
metabolic Pathway modification
The Mevalonate pathway was chosen for bioengineering modification as it is used by both A.annua and S.cerevisiae and the key enzymes and the intermediates responsible in production of artemisinic acid.
Biosynthesis of artemisinic acid involves using engineered Saccharomyces cerevisiae to produce high concentration of artemisinic acid. It is achieved by converting FPP to amorphadiene which subsequently oxidises by a cytochrome into artemisinic acid. (Ro et al., 2006)
To ensure the genetic stability; all of these modifications were made by chromosomal integration of the host yeast stain and performed in three steps;
Engineering the farnesyl pyrophosphate (FPP) biosynthetic pathway (MVA) to be over-expressed in order to increase FPP production while decreasing its use for ergosterol
Introducing the amorph-4, 11-adiene (amorphadiene) synthase gene (ADS) from A.annua to host yeast to convert high FPP production into amorphadiene
Cloning a cytochrome (P450) along with a CPR to performs a 3-step oxidation of amorphadiene to artemisinic acid from A.annua, when expressed in the host yeast strain (Ro et al., 2006)
Figure 2a- Schematic representation of engineered pathway in S.cerevisiae expressing A.annua genes (Ro et al., 2006)
Engineering modification of MVP pathway of S.cerevisiae can be performed by over-expressing the rate determining enzyme, HMGR, which is responsible for FPP conversion to sterol.
Such modification, even though improves the amorphadiene production, it however results decline in cell density due to insufficient production of ergosterol. As such, integration of an additional copy of HMGR gene into the S.cerevisaie chromosomal DNA should be conducted to increase amorphadiene production radically yet preventing decline in cell density.
The combined variation result in significantly high amount of amorphadiene and a sesquiterpene production, several folds higher than the yeast engineered with ADS alone. (Jackson et al., 2003)
Similarly, ERG9 gene which encodes for squalene synthase (SQS) can be down-regulated using methionine repressible promoter (PMET3) allowing the sterol production to be reduced while increasing the FPP conversion to amorphadiene by further two-folds. (Ro et al., 2006)
UPC2 is a global transcription factor that regulates biosynthesis of sterols in S. cerevisiae. Its activity is enhanced by a semi-dominant mutant allele (upc2-1). According to Davies et al., (2005), over-expressing of (upc2-1) has a mild effect on amorphadiene production. However, the combined variation in ERG9 (down-regulating) and upc2-1 (over-expressing) improves amorphadiene production by considerate amount. (Ro et al., 2006)
Monooxygenase (P450) is a conserved biosynthetic enzyme and is responsible for amorphadiene oxidation in A. annua. Its corresponding full-length cDNA sequence (CYP71AV1) can be recovered from A. annua DNA and introduced in S. cerevisiae under the control of a strong HXT7 promoter.
To have a functional heterologous expression of CYP71AV1; its redox partner, Cytochrome P450 oxidoreductase (CPR) must also be isolated from A. annua as shown below in Figure 1B.
Figure B- schematic representation of engineered genetic DNA sequence in a plasmid (Acosta et al., 2012)
A similar approach has been taken previously by Eelco Wallaart et al., 2000. Where cDNA clone encoding amorphadiene synthase was isolated from A.annua and expressed in E.coli. The recombinant enzyme demonstrated the conversion of amorphadiene to FDP.
To check whether or not CYP71AV1 is capable of catalysing all 3-step oxidation reactions; Ro et al., 2006 performed experimental observations in vitro where microsomes from engineered S. cerevisiae strain expressing CPR and CYP71AV1, were incubated along with pathway intermediates (amorphadiene, artemisinic alcohol and artemisinic aldehyde).
The experiment illustrated oxidation of all three intermediates to final product artemisinic acid. This demonstrates the ability and functionality of the recombinant cDNA at the C12 position of amorphadiene. (Bertia et al., 2005)
HmgR Phosphorylation inhibition
During metabolic pathways, HMGR is momentarily down-regulated by means of phosphorylation. In S. cerevisiae, phosphorylation of HMGR occurs at Serine 872 residue by AMP- activated protein kinase. Phosphorylation occurs when energy charge is low in the cell and there is a rise in AMP concentration. However HMGR is the rate determining step enzyme and is responsible for FPP conversion to sterol which subsequently converted to artemisinic acid. As such, HMGR phosphorylation would have detrimental effect in the production rate of artemisinic acid. (Goldstein & Brown, 1990)
To inhibit phosphorylation; site-specific mutagenesis can be performed on DNA sequence of HMGR gene. Serine 872 (AGA) can be replaced with Alanine (CGA). Phosphorylation cannot proceed, and structural conformation of protein will not occur. As a result, up-regulation of the artemisinic acid formation pathway can be achieved.
The insertion of bioengineered and heterologous genes (taken from A.annua) into S.cerevisiae, can be conducted via Molecular cloning which involves cloning of DNA fragments into a host organism through several mechanistic steps.
A cloning vector such as plasmid is used 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 during transformation. (Brown., 2006)
Genomic DNA that to be cloned is extracted from A.annua and purified from contaminating molecules using simple methods. Polymerase chain reaction or PCR is one of the methods, often used to amplify specific DNA sequences prior to molecular cloning.
The purified genomic DNA along with the modified mevalonate pathway sequence of S.cerevisiae are then treated with restriction endonuclease to generate fragments with ends compatible to those of the expression plasmid. To ensure a cleavage site that is compatible with that of the rDNA ends; both the expression plasmid DNA and rDNAs must be cleaved with the same restriction enzyme. (Russell & Sambrook., 2001)
Once completed, the transformation of S.cerevisiae can be carried out using electroporation which involves the use of high voltage electrical pulses to translocate DNA effectively across the cell membrane. Transformation is rarely 100%, only a small fraction of cells take up exogenous DNA. As a result, artificial genetic selection is used where cells that can actively replicate encoded DNA can be isolated using selectable marker gene.
Immobilisation is a well known technique used in many industrial applications to immobilise biological molecules/ living cells. In pharmaceutical industry, immobilisation usually carried out by entrapment of plant protoplasts, bacteria or enzymes in ionic network. Other methods of immobilisation are gelification, physical adsorption, ionic binding, covalent binding or cross-linking. (Giorno & Drioli., 2000)
One of the examples is the production of ampicillin and amoxicillin by entrapping penicillin Amidase in cellulose triacetate fibres by ionic network formation, which further demonstrates the practicality of entrapment technique 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 chain 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)
Entrapment can be performed by a single-step method, however the preparation usually suffer from heat damage and low mechanical strength. Alternatively, two-step methods may be employed which 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)
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. A suitable approach for production of small and porous beads under controlled conditions is necessary. Studies conducted by Ulku Mehmetoglu (1990) on immobilised S.cerevisiae cells by calcium alginate, suggests that the bead size of 1.75mm should be used as it gives good rate of reaction effective diffusion coefficient.
Advantages and Disadvantages
Free suspended yeast cells are sensitive to mechanical and fluid shear, as they can cause disruption. Feedstock cannot be transported using conventional pumps without significant loss of viability during pumping. Immobilisation provides solution to such problems as it protects yeast cells from sustained shear with no equipment modification. (Williams & Mavituna., 1992)
Physical separation of cells from the medium means simplicity of medium exchange or nutrient replenishment and product purity. The composition of the culture medium can be readily monitored via an external loop, and the concentrations of O2 or glucose can be adjusted as required. (Tyler et al., 1995)To avoid crabtree effect, the glucose feed should be no greater than 0.1%.
In terms of system stability, entrapment 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 because of the Ca+2 cross-links forming positively charged surfaces, suppressing Van der Waals attractive forces interacting between beads. On the other hand, repulsive forces become dominant keeping the beads suspended, as shown in Figure 5A below. The suspended system is said to high zeta potential, far away from their iso-electric point (i.e.p) where particles carry no net electrical charge and flocculate.
Figure 5A- Stable particle suspension (Greenwood, 2012)
One of the main disadvantages of immobilisation is that high productivity is only achieved by the cells suspended near the surface of the beads. Attempt to obtain the released artemisinic acid from the cells at the centre, is usually done by techniques such as permeabilisation which however decrease cells' viability to an undesirable extend. (Hutabarat, 2001)
Asexual reproduction of S.cervisiae is relatively short (about 90 minutes). Attempts to operate the process for extended period mean increase in cell density and physical rupture of the matrix. One way to tackle this problem is to ensure even distribution of cells in the beads. Also, to compensate space and nutrients with the newly born cells; fewer cells should be immobilised per bead initially.
Aeration of Immobilised Cells
Oxygen is a key nutrient in cellular metabolism. Low oxygen concentration affects cell growth and overall product yield, while oxygen in excessive amount can potentially be toxic due to superoxide radicals generation which may affect cells directly or indirectly by oxidising the medium component. Oxygen requirement of living cells varies according to different cell lines and culture conditions. Typical 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+2). 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 Roy et al, 2009 shows that a variation in chemical composition of the network can effectively control the swelling of the hydrophilic macromolecular matrices.
One can use higher amount of alginate to achieve a more compact matrix network. However this gives rise to smaller network pore sizes which consequently affects diffusional mass transfer of molecules cross the matrix.
Effect of pH and temperature
Alginate beads are said to be structurally stable at pH 4-4.5. Increase in pH affects their structural stability. Beyond pH 7.2, the COOH groups of alginate molecules undergo dissociation which weakens the physical forces between the two polymer chains, leading to bead disintegration and possibly flocculation as the zeta potential is decreased and the system is likely to reach its i.e.p.
Similarly temperatures higher than the optimum weakens the cross-links and attributes to the continuous erosion of surface layers of the beads which becomes faster with increasing temperature due to greater solubility.
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
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 and can be described by Fick's first law.
For internal mass balance, beads were modelled as porous catalyst spheres with cells being the active sites. Also, it was assumed that the reaction proceeds according to Michealis Menten kinetics, Ê‹ and that the liquid-film has no contribution at this stage. This gives a second order differential for glucose:
Replacing with, finally gives;
Note: Complete derivations for glucose, oxygen and artemisinic acid is given in the appendix.
Where DEff is the effective diffusion coefficient, dS is the change in concentration of glucose along the bead's radius, r.
Vmax represents the maximum rate achieved by the system at maximum substrate concentrations. Km is the Michaelis constant and represents the substrate concentration at half of Vmax
External mass transfer
Mass transport of nutrients and the removal of products from the bead surface, is usually occurred by convection and 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. As such, the external mass transfer for glucose is given in terms of boundary conditions.
kS - Average mass-transfer coefficient
S0 - Substrate concentrations in the bulk fluid
S* - Substrate concentration at solid-liquid interface.
As the liquid film at the interface was taken into account; the beads were considered as an uncharged, non-porous particle with the entire surface of which is uniformly accessible.
Note: Complete derivation is given in the appendix
Simultaneous mass transfer
Both the internal and external mass transfer resistances are significant and occur simultaneously. To obtain a combined analysis which consider both resistances and provide a dynamic model for substrate/product mass transport; the boundary conditions at each step were to be changed to dimensionless form.
Applying the dimensionless boundary conditions
The simultaneous mass transfer for substrate (glucose) gives;
Where, is the Thiele modulus - ratio of the intrinsic chemical reaction rate without mass transfer limitations, to the rate of diffusion through a particle. The Biot number, Bi, represents the relative degree to which the two resistances control mass transfer and is a function of liquid film thickness, diffusivity and the film mass transfer coefficient. This is because for a fluid flowing through a solid, the whole resistance to mass transfer is in the stagnant liquid film.
Note: Complete derivation is given in the appendix
The studies presented here provide a promising biosynthetic pathway to produce immediate drug precursor artemisinic acid by an engineered yeast strain. The engineering modification was performed in three levels:
Genome- Manipulating HMGR Serine residue with Alanine, introducing amorphadiene synthase gene (ADS) from A.annua to host yeast to convert high FPP production into amorphadiene and introducing a cytochrome to perform three step oxidation to artemisinic acid.
Proteome- inhibition of the down-regulation of HMGR by phosphorylation
Metabolome- overexpressed the biosynthetic MVA pathway to increase FPP production while decreasing its use for ergosterol.
Compared to A. annua, the engineered S. cerevisiae is expected to produce a much higher amount of artemisinic acid which is an immediate precursor of artemisinin, making biosynthesis cost-effective, fast and reliable source of immediate precursor artemisinin.
In terms of yeast cell immobilisation, even though gel bead entrapment suffers from mass transfer limitations; it is still far more practical method of immobilisation than the alternatives. One of the main alternatives is the immobilisation by natural cell floc formation, which although relatively cheaper and outperform entrapment, it still however far more problematic and challenging in terms of process stability, supply of energy mechanically, separation and purification.