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.Taxol (paclitaxel) and its structural analogs are among the most potent and commercially successful anticancer drugs (1). Taxol was first isolated from the bark of the Pacific yew tree (2), and early-stage production methods required sacrificing two to four fully grown trees to secure sufficient dosage for one patient (3). Taxol's structural complexity limited its chemical synthesis to elaborate routes that required 35 to 51 steps, with a highest yield of 0.4% (4-6). A semisynthetic route was later devised in which the biosynthetic intermediate baccatin III, isolated from plant sources, was chemically converted to Taxol (7). Although this approach and subsequent plant cell culture-based production efforts have decreased the need for harvesting the yew tree, production still depends on plant-based processes (8), with

A common strategy of metabolic engineering is to increase the endogenous supply of precursor metabolites to improve pathway productivity. The ability to further enhance heterologous production of a desired compound may be limited by the inherent capacity of the imported pathway to accommodate high precursor supply. Here, we present engineered diterpenoid biosynthesis as a case where insufficient downstream pathway capacity limits highlevel levopimaradiene production in Escherichia coli. To increase levopimaradiene synthesis, we amplified the flux toward isopentenyl diphosphate and dimethylallyl diphosphate precursors and reprogrammed the rate-limiting downstream pathway by generating combinatorial mutations in geranylgeranyl diphosphate synthase and levopimaradiene synthase. The mutant library contained pathway variants that not only increased diterpenoid production but also tuned the selectivity toward levopimaradiene. The most productive pathway, combining precursor flux amplification and mutant synthases, conferred approximately 2,600-fold increase in levopimaradiene levels. A maximum titer of approximately 700 mg∕L was subsequently obtained by cultivation in a benchscale bioreactor. The present study highlights the importance of engineering proteins along with pathways as a key strategy in achieving microbial biosynthesis and overproduction of pharmaceutical and chemical products.GGPP synthase | levopimaradiene synthase | metabolic engineering | Escherichia coli | molecular reprogramming M etabolic engineering is the enabling technology for the manipulation of organisms to synthesize high-value compounds of both natural and heterologous origin (1-4). In the case of heterologous production, well-characterized microorganisms are used as production hosts because targeted optimization can be performed using widely available genetic tools and synthetic biology frameworks (5, 6). One important application of engineered microbial systems is geared toward the synthesis of terpenoid natural products (7-9). Terpenoids represent one of the largest classes of secondary metabolites that includes pharmaceuticals, cosmetics, and potential biofuels candidates (8,(10)(11)(12). Metabolic engineering approaches to produce terpenoids in microbial systems such as Escherichia coli and yeast have commonly focused on increasing the precursor flux into the heterologous terpenoid pathway by rerouting endogenous isoprenoid metabolism (13-15). These engineering strategies have relied heavily on changing the enzyme concentrations in the product pathway.Many properties of a metabolic pathway, however, are not limited solely by the enzyme concentration, as is particularly true for the terpenoid pathway. In nature, terpenoid biosynthesis is regulated at multiple metabolic branch points to create large structural and functional diversity (16-18). In the major metabolic branch point in terpenoid biosynthesis, the prenyl transferases and terpenoid synthases catalyze the formation of a wide range of structurally diverse acyclic and cyclic terp...

BackgroundMicrobial production of lycopene, a commercially and medically important compound, has received increasing concern in recent years. Saccharomyces cerevisiae is regarded as a safer host for lycopene production than Escherichia coli. However, to date, the lycopene yield (mg/g DCW) in S. cerevisiae was lower than that in E. coli and did not facilitate downstream extraction process, which might be attributed to the incompatibility between host cell and heterologous pathway. Therefore, to achieve lycopene overproduction in S. cerevisiae, both host cell and heterologous pathway should be delicately engineered.ResultsIn this study, lycopene biosynthesis pathway was constructed by integration of CrtE, CrtB and CrtI in S. cerevisiae CEN.PK2. When YPL062W, a distant genetic locus, was deleted, little acetate was accumulated and approximately 100 % increase in cytosolic acetyl-CoA pool was achieved relative to that in parental strain. Through screening CrtE, CrtB and CrtI from diverse species, an optimal carotenogenic enzyme combination was obtained, and CrtI from Blakeslea trispora (BtCrtI) was found to have excellent performance on lycopene production as well as lycopene proportion in carotenoid. Then, the expression level of BtCrtI was fine-tuned and the effect of cell mating types was also evaluated. Finally, potential distant genetic targets (YJL064W, ROX1, and DOS2) were deleted and a stress-responsive transcription factor INO2 was also up-regulated. Through the above modifications between host cell and carotenogenic pathway, lycopene yield was increased by approximately 22-fold (from 2.43 to 54.63 mg/g DCW). Eventually, in fed-batch fermentation, lycopene production reached 55.56 mg/g DCW, which is the highest reported yield in yeasts.ConclusionsSaccharomyces cerevisiae was engineered to produce lycopene in this study. Through combining host engineering (distant genetic loci and cell mating types) with pathway engineering (enzyme screening and gene fine-tuning), lycopene yield was stepwise improved by 22-fold as compared to the starting strain. The highest lycopene yield (55.56 mg/g DCW) in yeasts was achieved in 5-L bioreactors. This study provides a good reference of combinatorial engineering of host cell and heterologous pathway for microbial overproduction of pharmaceutical and chemical products.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0509-4) contains supplementary material, which is available to authorized users.

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