Artemisinin

4. Metabolic Engineering

4.1 Isoprenoids

4.1.2 Artemisinin

Artemisinin, an endoperoxide sesquiterpene lactone, is an antimalarial drug produced in the leaves of Artemisia annua. Its biosynthesis comprises at least four enzymatic steps starting from FPP, the common precursor to sesquiterpenes. The first committed step is the cyclization of FPP to amorpha-4,11-diene, catalyzed by amorphadiene synthase (ADS). The cyctochrome P450 CYP71AV1 then oxidizes amorphadiene to artemisinic aldehyde and also makes artemisinic acid as a by-product.

Reduction to dihydroartemisinic aldehyde is catalyzed by artemisinic aldehyde ∆11 reductase, BDR2, and a specific aldehyde dehydrogenase is likely involved in the oxidation step to form hydroartemisinic acid, but the remaining steps in the pathway may be nonenzymatic.⁹³

The semisynthesis of artemisinin via metabolic engineering is based on the in vivo overproduction of amorphadiene or artemisinic acid and their subsequent conversion to artemisinin through chemical reactions (figure 16). Initial experiments aimed at improving the isoprenoid flux in E. coli focused on introducing the heterologous MVA pathway from S. cerevisiae to circumvent the regulatory elements found in E. coli’s native MEP pathway.⁸⁷ The recombinant eight-gene biosynthetic pathway was divided into two operons: a “top” operon (MevT) that encodes the three enzymes required to convert the ubiquitous precursor acetyl-CoA to (R)-mevalonate and a “bottom” operon (MBIS) that converts (R)-mevalonate to FPP (figure 16). Coexpression of these operons with a synthetic ADS gene, optimized for expression in E. coli, resulted in the production

of amorphadiene from glucose and glycerol with a final concentration of 0.5 g L⁻¹ following optimization of process conditions.^{87, 94}

Figure 16. Semisynthetic scheme for artemisinin production. Enzymes encoded by the genes shown are: atoB, acetoacetyl-CoA thiolase; HMGS, HMG-CoA synthase; tHMGR, truncated HMG-CoA reductase (expressing only the C-terminal catalytic domain); MK, mevalonate kinase; PMK, phosphomevalonate kinase; MPD, mevalonate diphosphate decarboxylase; idi, IPP isomerase; ispA, farnesyl diphosphate synthase; ADS, amorphadiene synthase; CPR, cytochrome P450 reductase; P450, CYP71AV1.⁹⁵

Mevalonate supplementation studies suggested that the production of amorphadiene in this system was limited by the conversion of acetyl-CoA to mevalonate.⁹⁶ However, increased expression of the three enzymes in the top operon (MevT) ⎯ by increasing plasmid copy number and by replacing the promoter ⎯ resulted in cell growth inhibition. Gene titration studies and metabolite profiling using LCMS

Acetyl -

CoA ^{atoB HMGS tHMGR} Mevalonate MK PMK MPD idi ispA FPP ADS Amorphadiene CPR P450 Artemisinic Acid Top MVA Pathway

(MevT) Bottom MVA Pathway

(MBIS) Synthase Hydroxylase Unit

S. cerevisiae A. annua

SCoA O

HO₂C HO

OH OPP

H

H

H

H O HO

H

H O O O

H H

O O In vivo biotransformations (E. coli or S. cerevisiae)

In vitro chemical transformations

H

H

H

HOH

H

OH

H

OH OH 1) 9-BBN

THF -78 °C 2) H₂O₂

NaOH

1) SO3 ⋅ pyridine 2) CH₂Cl₂, - 10 °C

1) NaOCl, NaH₂PO₄ DMSO, -10 °C

2) H3PO4, H2O Route A (amorphadiene → artemisinin)

H

H O HO

NaBH₄, NiCl₂ ⋅ 6H₂O Route B (artemisinic acid → artemisinin)

hν, O₂, CH₂Cl₂, - 78 °C

linked growth inhibition with the intracellular accumulation of the heterologous metabolite 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA), suggesting that low activity of a single enzyme, HMG-CoA reductase (tHMGR), encoded in MevT, was the major bottleneck. This flux imbalance was alleviated by coexpressing the MevT operon with an additional medium-copy-number plasmid carrying an extra copy of tHMGR, effectively increasing the concentration of the catalyst in the bottleneck step and thereby achieving a 3-fold increase in mevalonate production. Further optimizations included replacing the yeast genes for HMG-CoA synthase and HMG-CoA reductase with equivalent genes from Staphylococcus aureus, and improving the fermentation process under the restriction of carbon and nitrogen, so as to maximize flux toward the desired metabolic pathway, yielding commercially relevant titers of 27.4 g L^-1 amorphadiene.⁹⁷

The membrane-associated cytochrome P450 monooxygenase from A. annua (CYP71AV1) catalyzes the three-step oxidation of amorphadiene to artemisinic acid, producing the alcohol and aldehyde congeners of artemisinic acid as intermediates. To achieve the in vivo oxidation of amorphadiene to artemisinic acid, CYP71AV1 and its redox partner cytochrome P450 reductase (CPR), were codon-optimized for E. coli and coexpressed in the aforementioned strains. By engineering the N-terminal transmembrane sequence and using an expression plasmid suitable for P450 expression, artemisinic acid titers of 105 mg L^-1 were achieved, when conducting the fermentation in the absence of a n-dodecane overlay.⁹⁸ Conversion of artemisinic acid to the drug aremisinin can be accomplished in just two in vitro chemical reactions, namely reduction to hydroartemisinic acid followed by a photooxidation cyclization reaction (figure 16).⁹⁹

Biosynthesis of artemisinic acid has also been achieved in the yeast S.

cerevisiae, by up-regulating the expression of several genes from the native MVA pathway and by heterologous expression, through chromosomal integration, of amorphadiene synthase (ADS), amorphadiene monooxygenase (CYP71AV1) and the reductase (CPR).¹⁰⁰ Since steroid biosynthesis in yeast competes with ADS for FPP availability, it was necessary to down-regulate expression of squalene synthase, which catalyzes the first step downstream of FPP in the sterol pathway. Just as for E. coli, integration of an additional copy of tHMGR into the chromosome increased amorphadiene production, altogether producing >100 mg L^-1 artemisinic acid.

Interestingly, artemisinic acid was found to accumulate on the outer side of the cell membrane, allowing for a one-step purification following its “total” synthesis in yeast.

Artemisinic acid removed from the cell pellet by washing with alkaline buffer was extracted into ether and purified by silica gel chromatography to achieve more than 95%

purity.¹⁰⁰ Further strain engineering could not improve titers beyond 0.2 g L^-1 for glucose-fed fermentations.^95b However, strains that had been cloned with the pathway only up to amorphadiene achieved titers of more than 1.2 g L^-1. Subsequent improvements in the fermentation process for amorphadiene were able to significantly increase product titers up to the commercially relevant target of 37 g L^-1.^95b Key developments included restricting phosphate in fed-batch fermentations—this is thought to limit growth and channel greater carbon flux to product—and replacing the restricted glucose feed with a restricted ethanol feed, which is thought to increase intracellular acetyl-CoA levels for the MVA pathway. Deletion of GAL1 GAL10 GAL7 and Gal80p, a

gene cluster required for the utilization of galactose, altogether eliminated the need to add expensive galactose for induction of amorphadiene production. The S. cerevisiae–

based process has considerable advantages over the aforementioned E. coli process, most notably the absence of an expensive inducer such as IPTG and the lower oxygen uptake rate required for the restricted ethanol process, which reduces costs associated with aerating large fermentors.

The more facile biosynthesis of amorphadiene compared to artemisinic acid led to an alternative semisynthetic strategy where yeast fermentation converts sugars to amorphadiene and in vitro chemical reactions subsequently convert amorphadiene to dihydroartemisinic acid en route to artemisinin (figure 16).^95b Industrial production of semisynthetic artemisinin is being pursued by Amyris Biotechnologies in collaboration with Sanofi-Aventis.