Part:BBa_K1808010
ALDH1 generator
This device encodes for the production of the enzyme ALDH1. This device is an element of TCD iGEM 2015 set of parts for the synthesis of the anti-malarial drug precursor artemisinic acid in E. coli. The ALDH1 gene encodes the enzyme aldehyde dehydrogenase 1, which catalyses the conversion of (11R)-dihydroartemisinic aldehyde to dihydroartemisinic acid as well as the conversion of artemisinic aldehyde to artemisinic acid with NADP+ as the substrate. Artemisinic acid and dihydroartemisinic acid can be extracted from E. coli cells through lysis and chemically converted to the anti-malarial artemisinin. This part is a composite of the strong Anderson promoter (BBa_J23101), RBS (BBa_B0032), the coding region of the ALDH1 gene from Artemisia annua with a 6x His tag (BBa_K1808004) and a double terminator (BBa_B0015). The purpose of this device was to verify the activity of the aldehyde dehydrogenase1 enzyme in E. coli cells.
Usage and Biology
The potent antimalarial drug is naturally produced by the Artemisia annua plant that is native to Northern and Central China. Groundbreaking metabolic engineering work carried out by a team from UC Berkeley and Amyris Inc. led to the development of semi-synthetic artemisinin. Genes from the plant encoding one synthesis pathway were introduced into E. coli cells however, an alternative pathway involving the genes DBR2 and ALDH1 is also naturally present in the plant that was not discovered at the time of Amyris' work.
Trinity College Dublin 2015 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. The ALDH1 gene encodes the enzyme aldehyde dehydrogenase 1, which catalyses the conversion of (11R)-dihydroartemisinic aldehyde to dihydroartemisinic acid with NADP+ as the cofactor. The second purpose of ALDH1 is to catalyse the same step as the amorpadiene oxidase encoded by CyP71AV1 that is already present in B569 cells to increase the overall quantity of artemisininic acid produced by the cells, making them more efficient. An increased yield of the precursors would reduce the cost of the antimalarial drug, thus increasing access to it in the poorest affected countries, where often malaria is most endemic.
The aldehyde dehydrogenase1 enzyme is not specific to artemisinic aldehyde/dihydroartemisinic aldehyde but instead acts as a dehydrogenase on a large spectrum of aldehydes (mostly short chain aldehydes) including acetaldehyde. As such, the purpose of this construct was to exploit this feature and verify the function of the enzyme as synthesized in E. coli.
This construct overexpresses the ALDH1 gene due to the high copy number plasmid backbone used and the strong constitutive Anderson promoter. The RBS used exhibits only 30% efficiency when compared to BBa_B0034 - this is in order to decrease translation of mRNA and maintain host cell viability. Notably, this construct was used to verify the expression and activity of ALDH1. By itself, this construct is not enough to initiate an alternative artemisinin precursor synthesis pathway in B569 cells but theoretically should increase the efficiency of the final conversion step in the Amyris developed pathway.
Characterization
DH5-alpha cells were transformed with this construct to test for aldehyde dehydrogenase activity, taking advantage of the broad spectrum of activity. Cells expressing ALDH1 were lysed (freeze-thaw), the lysate spun down at 10K RPM for 10min and 40μl of the supernatant reacted with an acetaldehyde and cofactor solution. If ALDH1 enzymatic activity was present, acetaldehyde would be converted to acetic acid, which could subsequently be identified by running a GCMS analysis.
While DH5-alpha E. coli cells have been documented to contain a native acetaldehyde dehydrogenase enzyme, it is expressed in low levels generally and is only upregulated as part of a stress response if the cells are grown in ethanol containing media. For the assay, the cells were never exposed to ethanol and were lysed prior to addition to acetaldehyde solution, thus never allowing this upregulation to occur. In addition to this fact, the ALDH1 generator was in a high copy number plasmid and under the control of a strong constitutive promoter (BBa_J23101) - both factors signifying that ALDH1 would be strongly overexpressed in the transformed cell lines. As such, any native dehydrogenase activity could be negated by using the gentle freeze-thaw lysis and protein isolation techniques, only useful for working with overexpressed recombinant proteins as endogenous protein content is too low to be extracted by this manner.
The experiment was carried out in quadruplicate and GC-MS data showed a peak characteristic of the presence of acetic acid (RMatch values of ~820) in each case, confirming the activity of the dehydrogenase (see graphs a-d). No acetic acid was present in the reference standard meaning that the peak seen in the graphs is the result of recombinant dehydrogenase activity. It is therefore possible to extrapolate, based on these results that if artemisinic aldehyde/dihydroartemisinic aldehyde is present in the cell, aldehyde dehydrogenase1 would act on them converting them to artemisinic acid/dihydroartemisinic acid, as expected.
References
Paddon C. J. et al.; (2013) High-level semi-synthetic production of the potent antimalarial artemisinin; Nature 496, 528–532.
Yang K. et al.; (2015) The activity of the artemisinic aldehyde Δ11(13) reductase promoter is important for artemisinin yield in different chemotypes of Artemisia annua L.; Plant Molecular Biology 88(4); 325-340.
Johnson B. H., Hecht M. H.; (1994) Recombinant Proteins Can Be Isolated From E. Coli Cells Using Repeated Cycles Of Freezing And Thawing; Bio/Technology, 12, 1357-1360.
Ho K. K., Wiener H.; (2005) Isolation and Characterization of an Aldehyde Dehydrogenase Encoded by the aldB Gene of Escherichia coli; Journal of Bacteriology, 187(3), 1067–1073.
Teoh, K, Polichuk, D, Reed, D, & Covello, P 2009, 'Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemisia annua', Botany, 87, 6, pp. 635-642
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 83
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1332
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