Team:TrinityCollegeDublin/Design
E.artemisia Design
The focus of TCD iGEM 2015 team's "E. artemisia" project was the introduction of an alternative synthesis pathway for the production of a precursor of artemisinin, used in artemisinin combination therapies (ACTs) for the treatment of malaria. Artemisinin was originally isolated and produced solely from the Artemisia annua plant that is native to northern China and has a long history of being used in traditional medicines. However, supplies of and accessibility to the anti-malarial drug fluctuated greatly with yearly growth conditions as well as economic circumstances for farmers. Semi-synthetic artemisinin as developed by Amyris has led to the alleviation of this supply pressure by providing the means to synthesise the drug all year round and possibly reduce the price of treatment, making it available to more of those affected in the most susceptible, developing countries. Amyris succeeded in biologically producing artemisinic acid, which could be converted to artemisinin in a photoreactor.
Trinity iGEM intended to introduce an alternative synthesis pathway into the artemisinic acid producing E. coli cell line developed by Amyris. The alternative pathways was not known at the time that Amyris was developing its strains.
A total of seven different genes were designed by our team and synthesised by IDT with added BioBrick prefix and suffix, codon optimisation for expression in E. coli and synthesis as well as 6 His tag for prospective protein purification.
The seven genes were:
Our team proposed that a plasmid construct containing an operon for the expression of DBR2 and ALDH1 incorporated into Amyris' artemisinic acid producing E. coli B569 cells would allow the cells to produce a greater proportion of the more desirable intermediate dihydroartemisinic acid that is generated by the alternative pathway. Dihydroartemisinic acid requires less chemical conversion steps to be changed into artemisinin. In addition, the presence of ALDH1, which also catalyses some of the same steps as the amorpadiene oxidase encoded by CyP71AV1 that is already present in B569 cells, might increase the overall quantity of artemisinin precursors produced by the cells, making them more efficient. An increased yield would reduce the cost of the antimalarial drug, thus increasing access to it in the poorest affected countries, where often malaria is most endemic.
It was proposed by a number of research teams that a buildup of artemisinic aldehyde in E. coli cells is toxic yet it is necessary to have high levels of this intermediate for efficient production of artemisinic acid. The alternative pathway introduced by our team deviates from the original pathway at the point of this intermediate, with DBR2 using it up to generate dihydroartemisinic aldehyde thus relieving the pressure of the toxic intermediate on the cells. The ALDH1 enzyme would also serve the same role as it can act on the aldehyde to convert it to artemisinic acid.
Overall, Amyris made a decision to change from E. coli to yeast as a result of lower yields of artemisinic acid produced. With the addition of this alternative pathway, the yields from E. coli can be raised to higher, more economically viable levels. E. coli as the host organism for this pathway has several advantages over yeast, notably: it is easier and cheaper to grow and maintain, it grows faster and is easier to modify and it is more accessible as an organism for any company that wishes to produce semi-synthetic artemisinin in or closer to the countries where it is most needed.
An important consideration in our project was the Sanofi/Genzyme patent on yeast production of artemisinin that limits access to the technology for interested parties such as the charity Zagaya, which would otherwise produce artemisinin more openly and without the need for profits. Creating a viable E. coli cell-line for this purpose might be a means to bypass Sanofi's monopoly.
The assembly of the final construct (Alpha) from synthesised genes was simulated using Geneious R8 software. The method chosen for the assembly was a sequence of 3A protocols carried out to firstly attach a RBS to each part (and transfer to a Kanamycin resistance plasmid), followed by attachment of a promoter to DBR2 and a terminator to ALDH1 (and transfer to an Ampicillin resistance plasmid) and subsequent assembly of the two previous intermediates to form the final "Alpha" construct in a Kanamycin resistance plasmid. The plasmid backbones used had functional RFP generators which were removed during the digestion step. If, during the ligation, the RFP inserted ligated back into the plasmid backbone, the plasmid would express RFP and thus a colony that took up this wrong plasmid would appear red. White colonies would imply the correct ligation product was taken up by the cells. The "Alpha" construct, if introduced into B569 cells, should be able to initiate the alternative pathway.
Trinity iGEM intended to introduce an alternative synthesis pathway into the artemisinic acid producing E. coli cell line developed by Amyris. The alternative pathways was not known at the time that Amyris was developing its strains.
The seven genes were:
- DBR2 and ALDH1 - implicitly involved in the alternative synthesis pathway
- ADH1 and CyB5 - proposed to increase the efficiency of the existing Amyris developed pathway and
- ADS, CyP71AV1 and CPR - biobricked versions of important components of the Amyris developed pathway.
Our team proposed that a plasmid construct containing an operon for the expression of DBR2 and ALDH1 incorporated into Amyris' artemisinic acid producing E. coli B569 cells would allow the cells to produce a greater proportion of the more desirable intermediate dihydroartemisinic acid that is generated by the alternative pathway. Dihydroartemisinic acid requires less chemical conversion steps to be changed into artemisinin. In addition, the presence of ALDH1, which also catalyses some of the same steps as the amorpadiene oxidase encoded by CyP71AV1 that is already present in B569 cells, might increase the overall quantity of artemisinin precursors produced by the cells, making them more efficient. An increased yield would reduce the cost of the antimalarial drug, thus increasing access to it in the poorest affected countries, where often malaria is most endemic.
It was proposed by a number of research teams that a buildup of artemisinic aldehyde in E. coli cells is toxic yet it is necessary to have high levels of this intermediate for efficient production of artemisinic acid. The alternative pathway introduced by our team deviates from the original pathway at the point of this intermediate, with DBR2 using it up to generate dihydroartemisinic aldehyde thus relieving the pressure of the toxic intermediate on the cells. The ALDH1 enzyme would also serve the same role as it can act on the aldehyde to convert it to artemisinic acid.
Overall, Amyris made a decision to change from E. coli to yeast as a result of lower yields of artemisinic acid produced. With the addition of this alternative pathway, the yields from E. coli can be raised to higher, more economically viable levels. E. coli as the host organism for this pathway has several advantages over yeast, notably: it is easier and cheaper to grow and maintain, it grows faster and is easier to modify and it is more accessible as an organism for any company that wishes to produce semi-synthetic artemisinin in or closer to the countries where it is most needed.
An important consideration in our project was the Sanofi/Genzyme patent on yeast production of artemisinin that limits access to the technology for interested parties such as the charity Zagaya, which would otherwise produce artemisinin more openly and without the need for profits. Creating a viable E. coli cell-line for this purpose might be a means to bypass Sanofi's monopoly.
The assembly of the final construct (Alpha) from synthesised genes was simulated using Geneious R8 software. The method chosen for the assembly was a sequence of 3A protocols carried out to firstly attach a RBS to each part (and transfer to a Kanamycin resistance plasmid), followed by attachment of a promoter to DBR2 and a terminator to ALDH1 (and transfer to an Ampicillin resistance plasmid) and subsequent assembly of the two previous intermediates to form the final "Alpha" construct in a Kanamycin resistance plasmid. The plasmid backbones used had functional RFP generators which were removed during the digestion step. If, during the ligation, the RFP inserted ligated back into the plasmid backbone, the plasmid would express RFP and thus a colony that took up this wrong plasmid would appear red. White colonies would imply the correct ligation product was taken up by the cells. The "Alpha" construct, if introduced into B569 cells, should be able to initiate the alternative pathway.