In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 3 ( 2012-01-17)
Abstract:
This study links together the key steps of a complete production process for semisynthetic artemisinin. By combining high-level production of amorphadiene by fermentation of engineered S. cerevisiae with chemical conversion to dihydroartemisinic acid, we have achieved a significant milestone in the development of an economically viable process for the production of semisynthetic artemisinin. This process has the potential to significantly increase the supply of artemisinin for incorporation into ACTs and address the enormous problem of drug-resistant malaria in developing countries. This semisynthetic process for the production of artemisinin drastically reduces the time line of production and could increase the supply of artemisinin to meet world demand for supply of this drug. Our method can also make supplies available more quickly. Currently, it takes from 14 to 18 months from the time of planting to the extraction of artemisinin from the sweet wormwood plant, whereas a semisynthetic process could be accomplished in weeks ( 1 ) ( Fig. P1 ). Fig. P1. Production of plant-derived artemisinin compared to semisynthetic artemisinin. Production of plant-derived artemisinin takes from 14 to 18 months from planting to production. Plant-derived artemisinin requires cultivation of A. annua , followed by extraction of artemisinin from the leaves and conversion to artemisinin derivatives for incorporation into antimalarial ACT medication. Semisynthetic artemisinin, by contrast, uses engineered yeast to produce amorphadiene in fermentations. The amorphadiene extracted from the fermentor is chemically converted to dihydroartemisinic acid and then to artemisinin derivatives for incorporation into ACT drugs. The entire process could be accomplished in weeks. Producing amorphadiene by fermentation is only the first step in the production of semisynthetic artemisinin, because the amorphadiene still must be converted to artemisinin. In the plant A. annua , the first step to convert amorphadiene to artemisinin is the oxidation of amorphadiene, catalyzed by the cytochrome P450 enzyme, CYP71AV1 ( 5 ). The gene encoding this enzyme was isolated by finding homology to other, related cytochrome P450 enzymes. The gene was then functionally expressed (induced to produce its intended protein) in S. cerevisiae . This strain demonstrated that the enzyme could perform all three steps in the oxidation of amorphadiene to artemisinic acid ( 5 ). The work described in this report demonstrates production of artemisinic acid by the reengineered Gen 2.0 yeast, but at concentrations only one-tenth the concentration of amorphadiene. The reasons underlying this poor biological production of artemisinic acid were not investigated, but an alternative in vitro chemical procedure for the conversion of amorphadiene to dihydroartemisinic acid was developed; this conversion could conceivably be scaled to industrial levels. The conversion of dihydroartemisinic acid to artemisinin and its derivatives has been previously described. Previous engineering had been carried out in the prototypical laboratory yeast strain background S288C ( 5 ). We chose to engineer S. cerevisiae CEN.PK2 rather than S288C as its physiology is better studied, and its superior sporulation efficiency simplified strain engineering. CEN.PK2 was engineered in two different ways: ( i ) the engineering of the original S288C strain ( 5 ) was recapitulated (Gen 1.0 strain), and ( ii ) every enzyme of the biochemical pathway (termed the mevalonate pathway) required for the synthesis of farnesyl diphosphate (FPP), the amorphadiene precursor molecule, was overexpressed (induced to greater activity) (Gen 2.0 strain). The resulting strains showed striking differences in the production of amorphadiene and artemisinic acid. Overexpression of every mevalonate pathway enzyme required for FPP production (Gen 2.0) resulted in doubling of artemisinic acid production compared to the recapitulated strain (Gen 1.0), but a fivefold increase in amorphadiene production. The Gen 2.0 strain produced amorphadiene at a concentration that was tenfold higher than that of artemisinic acid; on the basis of this result, subsequent effort was directed at producing amorphadiene by using fed-batch fermentation. Manipulation of fermentation conditions, initially by restricting growth with phosphate limitation, then by the use of ethanol as a growth substrate, resulted in consistent production of over 30 g/L amorphadiene and maximal production of over 40 g/L. This paper describes the engineering of S. cerevisiae to produce amorphadiene at 40 g/L in fed-batch fermentation (a method that manages yeast growth by controlling the feeding of a certain nutrient). Initial work focused on eliminating the use of galactose, an expensive sugar, as the carbon source for growth, allowing the use of glucose, a cheaper sugar, as the growth substrate. Further strain engineering was then undertaken in an effort to increase production of both amorphadiene and its oxidized derivative, artemisinic acid. Increased demand for ACTs in the treatment of malaria has dramatically influenced the price and availability of artemisinin, an effective antimalarial drug extracted from the sweet wormwood plant ( Artemisia annua ). ACTs combine an artemisinin derivative with another antimalarial drug to reduce the chance that parasites will develop resistance to artemisinin ( 2 ). The increasing demand for this drug, along with limited supplies of plant-derived artemisinin ( 3 ), creates an urgent need to increase its supply. One potential solution, the production of amorphadiene, a precursor of artemisinin, has been previously demonstrated in both engineered Escherichia coli bacteria at 25 g/L ( 4 ) and S. cerevisiae , though at only 0.15 g/L ( 5 ). Malaria is caused by parasites of the Plasmodium species, primarily Plasmodium falciparum and Plasmodium vivax . Almost one million deaths from malaria and over 200 million new infections are recorded annually, primarily among young children in the developing world. In recent years, malaria parasites have developed resistance to all inexpensive drugs available in those areas, rendering these drugs ineffective. In response, the World Health Organization recommended the use of alternative treatments called artemisinin-based combination therapies (ACTs). This report describes progress toward the development of a semisynthetic production process whereby an artemisinin precursor is produced by engineered yeast ( Saccharomyces cerevisiae) in large-scale fermentation processes, and the precursor is then chemically converted to artemisinin ( 1 ).
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1110740109
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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