Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast.

Ashish Misra,Eddy C Agbo,Ganesh Sriram,Matthew F Conway,Anne M Derrick,Tabish M Qureshi,Bao Lige,Joseph Johnnie

doi:10.3389/fmicb.2013.00200

Abstract

Synthetic biology enables metabolic engineering of industrial microbes to synthesize value-added molecules. In this, a major challenge is the efficient redirection of carbon to the desired metabolic pathways. Pinpointing strategies toward this goal requires an in-depth investigation of the metabolic landscape of the organism, particularly primary metabolism, to identify precursor and cofactor availability for the target compound. The potent antimalarial therapeutic artemisinin and its precursors are promising candidate molecules for production in microbial hosts. Recent advances have demonstrated the production of artemisinin precursors in engineered yeast strains as an alternative to extraction from plants. We report the application of in silico and in vivo metabolic pathway analyses to identify metabolic engineering targets to improve the yield of the direct artemisinin precursor dihydroartemisinic acid (DHA) in yeast. First, in silico extreme pathway (ExPa) analysis identified NADPH-malic enzyme and the oxidative pentose phosphate pathway (PPP) as mechanisms to meet NADPH demand for DHA synthesis. Next, we compared key DHA-synthesizing ExPas to the metabolic flux distributions obtained from in vivo 13C metabolic flux analysis of a DHA-synthesizing strain. This comparison revealed that knocking out ethanol synthesis and overexpressing glucose-6-phosphate dehydrogenase in the oxidative PPP (gene YNL241C) or the NADPH-malic enzyme ME2 (YKL029C) are vital steps toward overproducing DHA. Finally, we employed in silico flux balance analysis and minimization of metabolic adjustment on a yeast genome-scale model to identify gene knockouts for improving DHA yields. The best strategy involved knockout of an oxaloacetate transporter (YKL120W) and an aspartate aminotransferase (YKL106W), and was predicted to improve DHA yields by 70-fold. Collectively, our work elucidates multiple non-trivial metabolic engineering strategies for improving DHA yield in yeast.

Highlights

Artemisinin-based combination therapy (ACT) is currently the most commonly used treatment for malaria (Weathers et al, 2006; Eastman and Fidock, 2009; Price and Douglas, 2009), an infectious disease that is widespread in regions of Africa, Asia, and South America (Feachem et al, 2010; O’Meara et al, 2010)
Strategizing how to produce dihydroartemisinic acid (DHA) from these substrates at high yield is a complex problem, solving which requires answering the following questions. (i) What is the theoretical maximal yield of DHA on glucose or galactose? (ii) Is this yield limited by the availability of reductant cofactors? (iii) Which configuration of pathways and fluxes favors a high yield? (iv) Do current DHA-synthesizing strains operate near or distantly from such a configuration? (v) Which minimal sets of genetic interventions can drastically improve the yield of a low DHA-producing strain? Below, we describe our computational analyses (ExPa analysis and minimization of metabolic adjustment (MOMA) analysis) and experimental investigations (13C MFA) toward answering these questions
THEORETICAL MAXIMAL YIELD OF DHA OR artemisinic acid (AA) ON GLUCOSE The value of the maximal yield of DHA on glucose depends on whether the reductant cofactors NADH and NADPH are considered in the analysis, and whether a distinction is made between the cofactors NADH and NADPH

Summary

Introduction

Artemisinin-based combination therapy (ACT) is currently the most commonly used treatment for malaria (Weathers et al, 2006; Eastman and Fidock, 2009; Price and Douglas, 2009), an infectious disease that is widespread in regions of Africa, Asia, and South America (Feachem et al, 2010; O’Meara et al, 2010). It is necessary to explore avenues for reliable production of artemisinin that offer this drug at the minimal possible cost to developing countries (Covello, 2008). To achieve this goal, researchers have demonstrated the synthesis of artemisinin precursors in microbes (Ro et al, 2006; Zhang et al, 2008; Westfall et al, 2012; Paddon et al, 2013) or plants (e.g., Zhang et al, 2011) engineered to express genes from the A. annua artemisinin pathway, thereby enabling conversion of endogenously produced FPP to artemisinin precursors. Chemical syntheses for artemisinin from starting materials ranging from natural www.frontiersin.org

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