US 201201078.93A1 (19) United States (2) Patent Application Publication (10) Pub. No.: US 2012/0107893 A1 Ajikumar et al. (43) Pub. Date: May 3, 2012 (54) MICROBIAL ENGINEERING FOR THE Related U.S. Application Data PRODUCTION OF CHEMICAL AND PHARMACEUTICAL PRODUCTS FROM THE (63) Continuation-in-part of application No. 12/943,477, ISOPRENOID PATHWAY filed on Nov. 10, 2010. (60) Provisional application No. 61/280,877, filed on Nov. (75) Inventors: Parayil K. Ajikumar, Cambridge, 10, 2009, provisional application No. 61/388,543, MA (US); Gregory filed on Sep. 30, 2010. Stephanopoulos, Winchester, MA (US); Too Heng Phon, Kent Vale Publication Classification (SG) (51) Int. Cl. CI2P 5/00 (2006.01) (73) Assignee: Massachusetts Institute of (52) U.S. Cl. ........................................................ 435/166 Technolechnology CambridgeCambridge. MAUs,MA (US e. ABSTRACT (21) Appl. No.: 13/249,388 The invention relates to the production of one or more terpe noids through microbial engineering, and relates to the manu (22) Filed: Sep. 30, 2011 facture of products comprising terpenoids. Patent Application Publication May 3, 2012 Sheet 1 of 24 US 2012/0107893 A1 q1:bla *... *… ---- , ~~~~. &#x3x3&############# Patent Application Publication May 3, 2012 Sheet 2 of 24 US 2012/0107893 A1 3.3. §§§ Fig.2b Patent Application Publication May 3, 2012 Sheet 3 of 24 US 2012/0107893 A1 : Patent Application Publication May 3, 2012 Sheet 4 of 24 US 2012/0107893 A1 §. 8, ºp. 7. § so speechinchMEpplomg Patent Application Publication May 3, 2012 Sheet 5 of 24 US 2012/0107893 A1 33 ?×ך?zzzzzzzzzzzzzzzzzzzzzzzzzzzzzzz& §§§ 3. cquitéd Ratsge twº §§§§?*************???? ???????????????? Patent Application Publication May 3, 2012 Sheet 6 of 24 US 2012/0107893 A1 ---, £ºstseata £xpressiº &gto: § 3. <> tº 2**** 2 㺠&gtºots ºctor. 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No. 12/943,477, filed on Nov. 10, 2010, [0008] The metabolic pathway for Taxol and its analogs which claims the benefit under 35 U.S.C. §119(e) of U.S. consists of an upstreamisoprenoid pathway that is native to E. Provisional Application Ser. No. 61/280,877, entitled coli, and a heterologous downstream terpenoid pathway “Microbial Engineering for the Production of Chemical and (FIG. 6). The upstream mevalonic acid (MVA) or methyl Pharmaceutical Products from Isoprenoid Pathway,” filed on erythritol phosphate (MEP) pathways can produce the two Nov. 10, 2009 and U.S. Provisional Application Ser. No. common building blocks, isopentenyl pyrophosphate (IPP) 61/388,543, entitled “Microbial to Engineering for the Pro and dimethylallyl pyrophosphate (DMAPP), from which duction of Chemical and Pharmaceutical Products from Iso Taxoland other isoprenoid compounds are formed.” Recent prenoid Pathway,” filed on Sep. 30, 2010, the entire disclo studies have highlighted the engineering of the above sures of which are incorporated by reference herein in their upstream pathways to support biosynthesis of heterologous entireties. isoprenoids such as lycopene and artemisinic acid.” The downstream taxadiene pathway has been reconstructed in E. GOVERNMENT INTEREST coli, but, to-date, titers have not exceeded 1.3 mg/L.” [0009] The above rational metabolic engineering [0002] This work was funded in part by the National Insti approaches focused on either the upstream (MVA or MEP) or tutes of Health under Grant Number 1-R01-GMO85323– the downstream terpenoid pathway, implicitly assuming that 01A1. The government has certain rights in this invention. modifications are additive, i.e. a linear behavior.” While this approach can yield moderate increases in flux, it gener FIELD OF THE INVENTION ally ignores non-specific effects, such as toxicity of interme [0003] The invention relates to the production of one or diate metabolites, cellular effects of the vectors used for more terpenoids through microbial engineering. expression, and hidden unknown pathways that may compete with the main pathway and divert flux away from the desired BACKGROUND OF THE INVENTION target. Combinatorial approaches can avoid such problems as they offer the opportunity to adequately sample the parameter [0004] Terpenoids are a class of compounds with diverse space and elucidate these complex non-linear interactions.” uses, including in fragrances, cosmetics, food products, phar 28,29.30 However, they require a high throughput screen, maceuticals, and other products. Thus, methods for their effi which is often not available for many desirable natural prod cient and cost effective production are needed. ucts.” Yet another class of pathway optimization methods [0005] For example, Taxol and its structural analogs have has explored the combinatorial space of different sources of been recognized as the most potent and commercially suc the heterologous genes comprising the pathway of interest.” cessful anticancer drugs introduced in the last decade." Taxol was first isolated from the bark of the Pacific Yew tree,” and Still dependent on a high throughput assay, these methods early stage production methods required sacrificing two to generally ignore the need for determining an optimal level of four fully grown trees to supply sufficient dosage for one expression for the individual pathway genes and, as such, patient.” Taxol's structural complexity necessitated a com have proven less effective in structuring an optimal pathway. plex chemical synthesis route requiring 35-51 steps with [0010] In the present work, as an example of aspects of the highest yield of 0.4%.” However, a semi-synthetic route invention, we focus on the optimal balancing between the was devised whereby the biosynthetic intermediate baccatin upstream, IPP-forming pathway with the downstream terpe III was first isolated from plant sources and was subsequently noid pathway of taxadiene synthesis. This is achieved by converted to Taxol.” While this approach and subsequent grouping the nine-enzyme pathway into two modules—a plant cell culture-based production efforts have decreased the four-gene, upstream, native (MEP) pathway module and a need for harvesting the yew tree, production still depends on two-gene, downstream, heterologous pathway to taxadiene plant-based processes” with accompanying limitations of (FIG. 1). Using this basic configuration, parameters such as productivity and scalability, and constraints on the number of the effect of plasmid copy number on cell physiology, gene Taxol derivatives that can be synthesized in search for more order and promoter strength in an expression cassette, and efficacious drugs.” chromosomal integration are evaluated with respect to their [0006] Methods for the production of terpenoids and effect on taxadiene production. This modular and multivari derivatives of these compounds, and methods of making able combinatorial approach allows us to efficiently sample products that comprise such compounds, are needed. the main parameters affecting pathway flux without the need for a high throughput screen. The multivariate search across SUMMARY OF THE INVENTION multiple promoters and copy numbers for each pathway mod ule reveals a highly non-linear taxadiene flux landscape with [0007] Recent developments in metabolic engineering and a global maximum exhibiting a 15,000 fold increase in taxa synthetic biology offer new possibilities for the overproduc diene production over the control, yielding 300 mg/L produc tion of complex natural products through more technically tion of taxadiene in small-scale fermentations.