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.
Keywords: artemisinin, metabolic engineering, metabolic pathway, extreme pathway, isotope labeling, metabolic flux analysis, flux balance analysis, minimization of metabolic adjustment
Citation: Misra A, Conway MF, Johnnie J, Qureshi TM, Derrick AM, Agbo EC and Sriram G (2013) Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast. Front. Microbiol. 4:200. doi: 10.3389/fmicb.2013.00200
Received: 25 April 2013; Accepted: 25 June 2013;
Published online: 26 July 2013.
Edited by:David Nielsen, Arizona State University, USA
Reviewed by:Stefan Junne, Technische Universität Berlin, Germany
Copyright: © 2013 Misra, Conway, Johnnie, Qureshi, Derrick, Agbo and Sriram. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.
*Correspondence: Ganesh Sriram, Department of Chemical and Biomolecular Engineering, University of Maryland, 1208D, Chemical and Nuclear Engineering Building 090, College Park, MD 20742, USA e-mail: firstname.lastname@example.org
†Present address: Ashish Misra, DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Mumbai, India; Matthew F. Conway, Department of Chemical Engineering, Columbia University, NewYork, NY, USA.