Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli

Abstract

Taxol (paclitaxel) is a potent anticancer drug first isolated from the Taxus brevifolia Pacific yew tree. Currently, cost-efficient production of Taxol and its analogs remains limited. Here, we report a multivariate-modular approach to metabolic-pathway engineering that succeeded in increasing titers of taxadiene--the first committed Taxol intermediate--approximately 1 gram per liter (~15,000-fold) in an engineered Escherichia coli strain. Our approach partitioned the taxadiene metabolic pathway into two modules: a native upstream methylerythritol-phosphate (MEP) pathway forming isopentenyl pyrophosphate and a heterologous downstream terpenoid-forming pathway. Systematic multivariate search identified conditions that optimally balance the two pathway modules so as to maximize the taxadiene production with minimal accumulation of indole, which is an inhibitory compound found here. We also engineered the next step in Taxol biosynthesis, a P450-mediated 5α-oxidation of taxadiene to taxadien-5α-ol. More broadly, the modular pathway engineering approach helped to unlock the potential of the MEP pathway for the engineered production of terpenoid natural products.

Fig. 1

Multivariate-modular approach for isoprenoid pathway optimization. To increase the flux through the upstream MEP pathway, we targeted reported enzymatic bottlenecks (dxs, idi, ispD, and ispF) (gray) for over-expression by an operon (dxs-idi-ispDF) (21). To channel the overflow flux from the universal isoprenoid precursors, IPP and DMAPP, toward Taxol biosynthesis, we constructed a synthetic operon of downstream genes GGPP synthase (G) and taxadiene synthase (T) (37). Both pathways were placed under the control of inducible promoters in order to control their relative gene expression. In the E. coli metabolic network, the MEP isoprenoid pathway is initiated by the condensation of the precursors glyceraldehyde-3 phosphate (G3P) and pyruvate (PYR) from glycolysis. The Taxol pathway bifurcation starts from the universal isoprenoid precursors IPP and DMAPP to form geranylgeranyl diphosphate, and then the taxadiene. The cyclic olefin taxadiene undergoes multiple rounds of stereospecific oxidations, acylations, and benzoylation to form the late intermediate Baccatin III and side chain assembly to, ultimately, form Taxol.

Fig. 2

Optimization of taxadiene production through regulating the expression of the upstream and downstream modular pathways. (A) Response in taxadiene accumulation to changes in upstream pathway strengths for constant values of the downstream pathway. (B) Dependence of taxadiene on the downstream pathway for constant levels of upstream pathway strength. (C) Taxadiene response from strains (17 to 24) engineered with high upstream pathway overexpressions (6 to 100 a.u.) at two different downstream expressions (31 a.u. and 61 a.u.). (D) Modulation of a chromosomally integrated upstream pathway by using increasing promoter strength at two different downstream expressions (31 a.u. and 61 a.u.). (E) Genotypes of the 32 strain constructs whose taxadiene phenotype is shown in Fig. 2, A to D. E, E. coli K12MG1655 ΔrecAΔendA; EDE3, E. coli K12MG1655 ΔrecAΔendA with DE3 T7 RNA polymerase gene in the chromosome; MEP, dxs-idi-ispDF operon; GT, GPPS-TS operon; TG, TS-GPPS operon; Ch1, 1 copy in chromosome; Trc, Trc promoter; T5, T5 promoter; T7, T7 promoter; p5, pSC101 plasmid; p10, p15A plasmid; and p20, pBR322 plasmid.

Fig. 3

Fed-batch cultivation of engineered strains in a 1-liter bioreactor. Time courses of (A) taxadiene accumulation, (B) cell growth, (C) acetic acid accumulation, and (D) total substrate (glycerol) addition for strains 22, 17, and 26 during 5 days of fed-batch bioreactor cultivation in 1-liter bioreactor vessels under controlled pH and oxygen conditions with minimal media and 0.5% yeast extract. After glycerol depletes to ~0.5 to 1 g/liter in the fermentor, 3 g/liter of glycerol was introduced into the bioreactor during the fermentation. Data are mean of two replicate bioreactors.

Fig. 4

Engineering Taxol P450 oxidation chemistry in E. coli. (A) TM engineering and construction of chimera protein from taxadien-5α-ol hydroxylase (T5αOH) and Taxus cytochrome P450 reductase (TCPR). The labels 1 and 2 represent the full-length proteins of T5αOH and TCPR identified with 42 and 74 amino acid TM regions, respectively, and 3 represents chimera enzymes generated from three different TM engineered T5αOH constructs [At8T5αOH, At24T5αOH, and At42T5αOH constructed by fusing an 8-residue synthetic peptide MALLLAVF (A) to 8, 24, and 42AA truncated T5αOH] through a translational fusion with 74AA truncated TCPR (tTCPR) by use of linker peptide GSTGS. (B) Functional activity of At8T5αOH-tTCPR, At24T5αOH-tTCPR, and At42T5αOH-tTCPR constructs transformed into taxadiene producing strain 26. Data are mean ± SD for three replicates. (C) Time course profile of taxadien-5α-ol accumulation and growth profile of the strain 26-At24T5αOH-tTCPR fermented in a 1-liter bioreactor. Data are mean of two replicate bioreactors.