“Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin” Westfall, P.J.; Pitera, D.J.; Lenihan, J.R.; Eng, D.; Woolard, F.X.; Regentin, R.; Horning, T.; Tsuruta, H.; Melis, D.J.; Owens, A.; Fickes, S.; Diola, D.; Benjamin, K.R.; Keasling, J.D.; Leavell, M.D.; McPhee, D.J.; Renninger, N.S.; Newman, J.D.; Paddon, C.J. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, E111-E118. DOI: 10.1073/pnas.1110740109.
Malaria, caused mainly by the parasite Plasmodium falciparum, leads to nearly a million deaths and 250 million new infections each year. The sesquiterpene lactone endoperoxide artemisinin, derived from Artemisia annua, is very effective as an antimalarial drug, and widespread resistance hasn’t yet developed. Artemisinin is the only high-volume drug that is still isolated by extraction from its native plant producer in a low-yielding (around 10 μg per g plant material), resource-intensive process that uses volatile solvents (most commonly hexane).
As a result, supplies of the drug are short, and those who need it often can’t afford it. The development of new processes for artemisinin production would therefore advance both public health and green chemistry interests. Total synthesis of the drug hasn’t been considered as a viable alternative because of low yields, but a lot of effort has been directed toward developing semi-synthetic sources of artemisinin using a combination of microbial fermentation and chemical synthesis. Toward this end, the Keasling lab reported a few years ago that they had constructed a biosynthetic pathway for the artemisinin precursor amorpha-4,11-diene in yeast with yields of ~200 mg/L—already impressive given the complexity of the molecule. Amorphadiene synthase (ADS) comes from Artemisia annua; the rest of the genes are from yeast. Here is the existing pathway:
A recent paper from Amyris Biotechnologies reports optimization of this pathway for industrial production to yield 40 g/L of amorpha-4,11-diene along with a chemical process that completes the production of semi-synthetic artemisinin.
The first step was to increase flux through the pathway by tuning gene expression. While previous strains had only overexpressed HMG-CoA reductase from the mevalonate pathway that leads to the amorpha-4,11-diene precursor farnesyl pyrophosphate, the researchers at Amyris elected to construct new strains that instead overexpress every gene leading to farnesyl pyrophosphate by placing them under strong, galactose-regulated promoters. Because expression of HMG-CoA reductase (tHMG1 in the above figure), which catalyzes the first committed step in the pathway, had previously been shown to be limiting, they also added two additional copies of the gene. These adjustments in gene expression led to a fivefold increase in amorpha-4,11-diene production.
Low yields are not the only limiting factor for commercialization of pathways from academic labs. Some of the strategies that are often used during initial pathway construction to allow high-level production of foreign proteins in microbial hosts add extra expense and complexity that is undesirable for economical large-scale production. One such problem that Amyris needed to overcome was that the original pathway was set up such that the presence of galactose was required for expression of the pathway genes. The galactose-requiring expression system adds a specific DNA sequence in front of foreign genes that is normally found in front of the genes required for yeast to grow on galactose. This sequence is recognized by a DNA-binding protein, Gal4, which also binds to some of the cellular machinery needed for transcription, ultimately leading to high levels of protein production. In the absence of galactose, a galactose-sensing protein, Gal80, binds to Gal4, blocking its interaction with the transcription machinery and shutting down protein production. This system is popular in metabolic engineering because it provides a convenient way to trick yeast into expressing any protein you want by co-opting the DNA sequence that controls expression of galactose breakdown genes. The drawback, however, is it requires the use of galactose as a carbon source, and that’s too expensive to be used in industrial-scale fermentations, even when yields are higher.
To get around the need to use galactose as a carbon feedstock while still taking advantage of the high levels of gene expression that Gal4 can promote, the scientists at Amyris deleted Gal80, the protein that blocks interaction of Gal4 with the transcription machinery in the absence of galactose. Without Gal80 to interfere with transcription, HMG-CoA reductase and amorphadiene synthase are expressed at high levels regardless of the carbon source. This modification produced a yeast strain that made amorpha-4,11-diene at similar levels to the original, but using glucose, which is cheap enough for large-scale applications, as a carbon source.
Fermentation conditions were then optimized for this strain. To direct carbon flux away from biomass accumulation (i.e., cell growth) and toward synthesis of amorpha-4,11-diene, phosphate levels in the yeast media were limited, increasing titers to 5.5 g/L. Based on the ability of yeast to convert ethanol to acetyl-CoA, the yeast were fed with mixtures of ethanol and glucose in an attempt to increase the cytosolic concentration of acetyl-CoA. This indeed increased yields significantly, to 16.5 g/L. Even better results were obtained by feeding with pure ethanol, which increased yields to the reported 40 g/L, corresponding to an impressive 20% yield from carbon.
Finally, to bridge the gap between amorpha-4,11-diene and artemisinin, they developed a synthetic strategy to convert amorpha-4,11-diene to dihydroartemisinic acid, the precursor of artemisinin.
Use of 9-borabicyclo[3.3.1]nonane (9-BBN) allowed selective hydroboration of the exocyclic double bond, giving dihydroartemisinic alcohol as an 85:15 mixture of epimers, with the desired epimer making up the bigger portion of the mixture. The researchers opted for a two-step oxidation to make the carboxylic acid, avoiding the use of toxic chromium-based oxidants and undesired reactivity of the double bond. Dihydroartemisinic alcohol was therefore oxidized to dihydroartemisinic aldehyde with sulfur trioxide-pyridine, and the aldehyde was taken to the acid with sodium chlorite in DMSO to give dihydroartemisinic acid in 48% overall yield from amorpha-4,11-diene. Luckily, the step that looks the most difficult to achieve synthetically, conversion of dihydroartemisinic acid to artemisinin, is believed to occur non-enzymatically in nature, requiring only molecular oxygen and light, so it seems very feasible to produce artemisinin itself using this synthesis, although, disappointingly, this was not explored in the paper. It will be exciting to see how the pathway is completed for industrial production.
As Courtney pointed out in his post on microbial butanediol production, a lot of papers from academic labs on microbial chemical production seem too low-yielding to ever be commercialized. This paper provides a great example in which what started out as a low-yielding pathway is now poised to change the way an important pharmaceutical is produced. The first doses of semi-synthetic artemisinin, which presumably will be produced by a pathway similar to the one developed in this paper, are slated to become available later this year through a partnership between Amyris, The Institute for OneWorld Health, and Sanofi-Aventis. In addition to addressing a public health concern and providing a platform for greener production of artemisinin, the yeast strains and methods developed in this paper may also have an impact on the production of many other chemicals. Only the final enzymatic step is specific to production of amorpha-4,11-diene, so it’s possible that the same yeast strain could easily be engineered for production of other terpene-derived molecules like flavors, fragrances, and advanced biofuels.