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dc.contributor.authorChen, Xixian
dc.contributor.authorZhang, Congqiang
dc.contributor.authorZou, Ruiyang
dc.contributor.authorZhou, Kang
dc.contributor.authorStephanopoulos, Gregory
dc.contributor.authorToo, Heng Phon
dc.date.accessioned2014-01-06T19:38:15Z
dc.date.available2014-01-06T19:38:15Z
dc.date.issued2013-11
dc.date.submitted2013-05
dc.identifier.issn1932-6203
dc.identifier.urihttp://hdl.handle.net/1721.1/83517
dc.description.abstractIn vitro synthesis of chemicals and pharmaceuticals using enzymes is of considerable interest as these biocatalysts facilitate a wide variety of reactions under mild conditions with excellent regio-, chemo- and stereoselectivities. A significant challenge in a multi-enzymatic reaction is the need to optimize the various steps involved simultaneously so as to obtain high-yield of a product. In this study, statistical experimental design was used to guide the optimization of a total synthesis of amorpha-4,11-diene (AD) using multienzymes in the mevalonate pathway. A combinatorial approach guided by Taguchi orthogonal array design identified the local optimum enzymatic activity ratio for Erg12:Erg8:Erg19:Idi:IspA to be 100:100:1:25:5, with a constant concentration of amorpha-4,11-diene synthase (Ads, 100 mg/L). The model also identified an unexpected inhibitory effect of farnesyl pyrophosphate synthase (IspA), where the activity was negatively correlated with AD yield. This was due to the precipitation of farnesyl pyrophosphate (FPP), the product of IspA. Response surface methodology was then used to optimize IspA and Ads activities simultaneously so as to minimize the accumulation of FPP and the result showed that Ads to be a critical factor. By increasing the concentration of Ads, a complete conversion (~100%) of mevalonic acid (MVA) to AD was achieved. Monovalent ions and pH were effective means of enhancing the specific Ads activity and specific AD yield significantly. The results from this study represent the first in vitro reconstitution of the mevalonate pathway for the production of an isoprenoid and the approaches developed herein may be used to produce other isopentenyl pyrophosphate (IPP)/dimethylallyl pyrophosphate (DMAPP) based products.en_US
dc.description.sponsorshipSingapore-MIT Allianceen_US
dc.language.isoen_US
dc.publisherPublic Library of Scienceen_US
dc.relation.isversionofhttp://dx.doi.org/10.1371/journal.pone.0079650en_US
dc.rights.urihttp://creativecommons.org/licenses/by/2.5/en_US
dc.sourcePLoSen_US
dc.titleStatistical Experimental Design Guided Optimization of a One-Pot Biphasic Multienzyme Total Synthesis of Amorpha-4,11-dieneen_US
dc.typeArticleen_US
dc.identifier.citationChen, Xixian, Congqiang Zhang, Ruiyang Zou, Kang Zhou, Gregory Stephanopoulos, and Heng Phon Too. “Statistical Experimental Design Guided Optimization of a One-Pot Biphasic Multienzyme Total Synthesis of Amorpha-4,11-diene.” Edited by Vishal Shah. PLoS ONE 8, no. 11 (November 20, 2013): e79650.en_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Chemical Engineeringen_US
dc.contributor.departmentSingapore-MIT Alliance in Research and Technology (SMART)en_US
dc.contributor.mitauthorChen, Xixianen_US
dc.contributor.mitauthorZhang, Congqiangen_US
dc.contributor.mitauthorZhou, Kangen_US
dc.contributor.mitauthorStephanopoulos, Gregoryen_US
dc.relation.journalPLoS ONEen_US
dc.eprint.versionFinal published versionen_US
dc.type.urihttp://purl.org/eprint/type/JournalArticleen_US
eprint.statushttp://purl.org/eprint/status/PeerRevieweden_US
dspace.orderedauthorsChen, Xixian; Zhang, Congqiang; Zou, Ruiyang; Zhou, Kang; Stephanopoulos, Gregory; Too, Heng Phonen_US
dc.identifier.orcidhttps://orcid.org/0000-0001-6909-4568
mit.licensePUBLISHER_CCen_US


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