James Kirby

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

Malaria is a global health problem that threatens 300–500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l-1) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices.

Biosynthesis of Plant Isoprenoids: Perspectives for Microbial Engineering

Isoprenoids are a large and highly diverse group of natural products with many functions in plant primary and secondary metabolism. Isoprenoids are synthesized from common prenyl diphosphate precursors through the action of terpene synthases and terpene-modifying enzymes such as cytochrome P450 monooxygenases. Many isoprenoids have important applications in areas such as human health and nutrition, and much effort has been directed toward their production in microbial hosts. However, many hurdles must be overcome in the elucidation and functional microbial expression of the genes responsible for biosynthesis of an isoprenoid of interest. Here, we review investigations into isoprenoid function and gene discovery in plants as well as the latest advances in isoprenoid pathway engineering in both plant and microbial hosts.

Redirection of Flux Through the FPP Branch-Point in Saccharomyces cerevisiae by Down-Regulating Squalene Synthase

Saccharomyces cerevisiae utilizes several regulatory mechanisms to maintain tight control over the intracellular level of farnesyl diphosphate (FPP), the central precursor to nearly all yeast isoprenoid products. High‐level production of non‐native isoprenoid products requires that FPP flux be diverted from production of sterols to the heterologous metabolic reactions. To do so, expression of the gene encoding squalene synthase (ERG9), the first committed step in sterol biosynthesis, was down‐regulated by replacing its native promoter with the methionine‐repressible MET3 promoter. The intracellular levels of FPP were then assayed by expressing the gene encoding amorphadiene synthase (ADS) and converting the FPP to amorphadiene. Under certain culture conditions amorphadiene production increased fivefold upon ERG9 repression. With increasing flux to amorphadiene, squalene and ergosterol production each decreased. The levels of these three metabolites were dependent not only upon the level of ERG9 repression, but also the timing of its repression relative to the induction of ADS and genes responsible for enhancing flux to FPP. Biotechnol. Bioeng. 2008;99: 371–378.

Engineering triterpene production in Saccharomyces cerevisiae–β-amyrin synthase from Artemisia annua

Using a degenerate primer designed from triterpene synthase sequences, we have isolated a new gene from the medicinal plant Artemisia annua. The predicted protein is highly similar to β‐amyrin synthases (EC 5.4.99.–), sharing amino acid sequence identities of up to 86%. Expression of the gene, designated AaBAS, in Saccharomyces cerevisiae, followed by GC/MS analysis, confirmed the encoded enzyme as a β‐amyrin synthase. Through engineering the sterol pathway in S. cerevisiae, we explore strategies for increasing triterpene production, using AaBAS as a test case. By manipulation of two key enzymes in the pathway, 3‐hydroxy‐3‐methylglutaryl‐CoA reductase and lanosterol synthase, we have improved β‐amyrin production by 50%, achieving levels of 6 mg·L−1 culture. As we have observed a 12‐fold increase in squalene levels, it appears that this strain has the capacity for even higher β‐amyrin production. Options for further engineering efforts are explored.

Anaerobic Central Metabolic Pathways in Shewanella oneidensis MR-1 Reinterpreted in the Light of Isotopic Metabolite Labeling

It has been proposed that during growth under anaerobic or oxygen-limited conditions, Shewanella oneidensis MR-1 uses the serine-isocitrate lyase pathway common to many methylotrophic anaerobes, in which formaldehyde produced from pyruvate is condensed with glycine to form serine. The serine is then transformed through hydroxypyruvate and glycerate to enter central metabolism at phosphoglycerate. To examine its use of the serine-isocitrate lyase pathway under anaerobic conditions, we grew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source, with either trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor. Analysis of cellular metabolites indicated that a large percentage (>70%) of lactate was partially oxidized to either acetate or pyruvate. The 13C isotope distributions in amino acids and other key metabolites indicate that under anaerobic conditions, although glyoxylate synthesized from the isocitrate lyase reaction can be converted to glycine, a complete serine-isocitrate pathway is not present and serine/glycine is, in fact, oxidized via a highly reversible degradation pathway. The labeling data also suggest significant activity in the anapleurotic (malic enzyme and phosphoenolpyruvate carboxylase) reactions. Although the tricarboxylic acid (TCA) cycle is often observed to be incomplete in many other anaerobes (absence of 2-oxoglutarate dehydrogenase activity), isotopic labeling supports the existence of a complete TCA cycle in S. oneidensis MR-1 under certain anaerobic conditions, e.g., TMAO-reducing conditions.

Production and quantification of sesquiterpenes in Saccharomyces cerevisiae , including extraction, detection and quantification of terpene products and key related metabolites

The procedures described here are designed for engineering Saccharomyces cerevisiae to produce sesquiterpenes with an aim to either increase product titers or to simply generate a quantity of product sufficient for identification and/or downstream experimentation. Engineering high-level sesquiterpene production in S. cerevisiae often requires iterations of strain modifications and metabolite analysis. To address the latter, the methods described here were tailored for robust measurement of metabolites that we have found to be fundamental indicators of pathway flux, using only gas chromatography and mass spectrometry (GC-MS) instrumentation. Thus, by focusing on heterologous production of sesquiterpenes via the mevalonate (MEV) pathway in S. cerevisiae, we detail procedures for extraction and detection of the key pathway metabolites MEV, squalene and ergosterol, as well as the farnesyl pyrophosphate (FPP)-derived side products farnesol and nerolidol. Analysis of these compounds is important for quality control, because they are possible indicators of pathway imbalance. As many of the sesquiterpene synthase (STS) genes encountered in nature are of plant origin and often not optimal for expression in yeast, we provide guidelines for designing gene expression cassettes to enable expression in S. cerevisiae. As a case study for these protocols, we have selected the sesquiterpene amorphadiene, native to Artemisia annua and related plants. The analytical steps can be completed within 1–2 working days, and a typical experiment might take 1 week.

Functional characterization of four sesquiterpene synthases from Ricinus communis (Castor bean)

Genome sequence analysis of Ricinus communis has indicated the presence of at least 22 putative terpene synthase (TPS) genes, 13 of which appear to encode sesquiterpene synthases (SeTPSs); however, no SeTPS genes have been isolated from this plant to date. cDNAs were recovered for six SeTPS candidates, and these were subjected to characterization in vivo and in vitro. The RcSeTPS candidates were expressed in either Escherichia coli or Saccharomyces cerevisiae strains with engineered sesquiterpene biosynthetic pathways, but only two (RcSeTPS1 and RcSeTPS7) produced detectable levels of product. In order to check whether the engineered microbial hosts were adequately engineered for sesquiterpene production, a selection of SeTPS genes was chosen from other plant species and demonstrated consistently high sesquiterpene titers. Activity could be demonstrated in vitro for two of the RcSeTPS candidates (RcSeTPS5 and RcSeTPS10) that were not observed to be functional in our microbial hosts. RcSeTPS1 produced two products, (-)-α-copaene and (+)-δ-cadinene, while RcSeTPS7 produced a single product, (E, E)-α-farnesene. Both RcSeTPS5 and RcSeTPS10 produced multiple sesquiterpenes.

Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

ABSTRACT Terpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5 sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of a dxs deletion in Escherichia coli grown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-type E. coli yajO gene, annotated as a putative xylose reductase, or via various mutations in the native ribB gene. In vitro analysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway in E. coli for production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

Use of nonionic surfactants for improvement of terpene production in Saccharomyces cerevisiae.

ABSTRACT To facilitate enzyme and pathway engineering, a selection was developed for improved sesquiterpene titers in Saccharomyces cerevisiae. α-Bisabolene, a candidate advanced biofuel, was found to protect yeast against the disruptive action of nonionic surfactants such as Tween 20 (T20). An experiment employing competition between two strains of yeast, one of which makes twice as much bisabolene as the other, demonstrated that growth in the presence of T20 provided sufficient selective pressure to enrich the high-titer strain to form 97% of the population. Following this, various methods were used to mutagenize the bisabolene synthase (BIS) coding sequence, coupled with selection by subculturing in the presence of T20. Mutagenesis targeting the BIS active site did not yield an improvement in bisabolene titers, although mutants were found which made a mixture of α-bisabolene and β-farnesene, another candidate biofuel. Based on evidence that the 3′ end of the BIS mRNA may be unstable in yeast, we randomly recoded the last 20 amino acids of the enzyme and, following selection in T20, found a variant which increased specific production of bisabolene by more than 30%. Since T20 could enrich a mixed population, efficiently removing strains that produced little or no bisabolene, we investigated whether it could also be applied to sustain high product titers in a monoculture for an extended period. Cultures grown in the presence of T20 for 14 days produced bisabolene at titers up to 4-fold higher than cultures grown with an overlay of dodecane, used to sequester the terpene product, and 20-fold higher than cultures grown without dodecane.

Development of an integrated approach for α-pinene recovery and sugar production from loblolly pine using ionic liquids

In the southeastern US, loblolly pine (Pinus taeda L.) is widely used as a feedstock in the wood, pulp and paper industry. In loblolly pine, the oleoresin is composed of terpenes and has long been a valuable source for a variety of chemicals, and has recently attracted interest from a biofuel perspective for the production of advanced cellulosic biofuels. To date, there have been very few examples where a single conversion process has enabled recovery of both terpenes and fermentable sugars in an integrated fashion. We have used the ionic liquid (IL), 1-ethyl-3-methylimidazolium acetate [C2C1Im][OAc] at 120 °C and 160 °C in conjunction with analytical protocols using GC-MS, to extract α-pinene and simultaneously pretreat the pine to generate high yields of fermentable sugars after saccharification. Compared to solvent extraction, the IL process enabled higher recovery rates for α-pinene, from three tissues type of loblolly pine, i.e. pine chips from forest residues (FC), stems from young pine (YW) and lighter wood (LW), while also generating high yields of fermentable sugars following saccharification. We propose that this combined terpene extraction/lignocellulose pretreatment approach may provide a compelling model for a biorefinery, reducing costs and increasing commercial viability. Our preliminary techno-economic analysis (TEA) revealed that the α-pinene recovery based on hexane extraction after IL pretreatment could reduce the minimum ethanol selling price (MESP) of ethanol generated from fermentation of sugars recovered from pine by