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

Engineering robust microbes for the biotech industry typically requires high-level, genetically stable expression of heterologous genes and pathways. Although plasmids have been used for this task, fundamental issues concerning their genetic stability have not been adequately addressed. Here we describe chemically inducible chromosomal evolution (CIChE), a plasmid-free, high gene copy expression system for engineering Escherichia coli. CIChE uses E. coli recA homologous recombination to evolve a chromosome with approximately 40 consecutive copies of a recombinant pathway. Pathway copy number is stabilized by recA knockout, and the resulting engineered strain requires no selection markers and is unaffected by plasmid instabilities. Comparison of CIChE-engineered strains with equivalent plasmids revealed that CIChE improved genetic stability approximately tenfold and growth phase-specific productivity approximately fourfold for a strain producing the high metabolic burden-biopolymer poly-3-hydroxybutyrate. We also increased the yield of the nutraceutical lycopene by 60%. CIChE should be applicable in many organisms, as it only requires having targeted genomic integration methods and a recA homolog.

Terpenoids represent a diverse class of molecules that provide a wealth of opportunities to address many human health and societal issues. The expansive array of structures and functionalities that have been evolved in nature provide an excellent pool of molecules for use in human therapeutics. While this class of molecules has members with therapeutic properties including anticancer, antiparasitic, antimicrobial, antiallergenic, antispasmodic, antihyperglycemic, anti-inflammatory, and immunomodulatory properties, supply limitations prevent the large scale use of some molecules. Many of these molecules are only found in ppm levels in nature thus requiring massive harvesting to obtain sufficient amounts of the drug. Synthetic biology and metabolic engineering provide innovative approaches to increase the production of the desired molecule in the native organism, and most importantly, transfer the biosynthetic pathways to other hosts. Microbial systems are well studied, and genetic manipulations allow the optimization of microbial metabolisms for the production of common terpenoid precursors. Using a host of tools, unprecedented advancements in the large scale production of terpenoids have been achieved in recent years. Identification of limiting steps and pathway regulation, coupled with design strategies to minimize terpenoid byproducts wih a high flux to the desired biosynthetic pathways, have yielded greater than 100-fold improvements in the production of a range of terpenoids. This review focuses on the biodiversity of terpenoids, the biosynthetic pathways involved, and engineering efforts to maximize the production through these pathways.

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...

Conversion of carbohydrates to lipids at high yield and productivity is essential for cost-effective production of renewable biodiesel. Although some microorganisms can convert sugars to oils, conversion yields and rates are typically low due primarily to allosteric inhibition of the lipid biosynthetic pathway by saturated fatty acids. By reverse engineering the mammalian cellular obese phenotypes, we identified the delta-9 stearoyl-CoA desaturase (SCD) as a rate limiting step and target for the metabolic engineering of the lipid synthesis pathway in Yarrowia lipolytica. Simultaneous overexpression of SCD, Acetyl-CoA carboxylase (ACC1), and Diacylglyceride acyl-transferase (DGA1) in Y. lipolytica yielded an engineered strain exhibiting highly desirable phenotypes of fast cell growth and lipid overproduction including high carbon to lipid conversion yield (84.7% of theoretical maximal yield), high lipid titers (~55g/L), enhanced tolerance to glucose and cellulose-derived sugars. Moreover, the engineered strain featured a three-fold growth advantage over the wild type strain. As a result, a maximal lipid productivity of ~1g/L/h is obtained during the stationary phase. Furthermore, we showed that the engineered yeast required cytoskeleton remodeling in eliciting the obesity phenotype. Altogether, our work describes the development of a microbial catalyst with the highest reported lipid yield, titer and productivity to date. This is an important step towards the development of an efficient and cost-effective process for biodiesel production from renewable resources.

Industrial biotechnology promises to revolutionize conventional chemical manufacturing in the years ahead, largely owing to the excellent progress in our ability to re-engineer cellular metabolism. However, most successes of metabolic engineering have been confined to over-producing natively synthesized metabolites in E. coli and S. cerevisiae. A major reason for this development has been the descent of metabolic engineering, particularly secondary metabolic engineering, to a collection of demonstrations rather than a systematic practice with generalizable tools. Synthetic biology, a more recent development, faces similar criticisms. Herein, we attempt to lay down a framework around which bioreaction engineering can systematize itself just like chemical reaction engineering. Central to this undertaking is a new approach to engineering secondary metabolism known as ‘multivariate modular metabolic engineering’ (MMME), whose novelty lies in its assessment and elimination of regulatory and pathway bottlenecks by re-defining the metabolic network as a collection of distinct modules. After introducing the core principles of MMME, we shall then present a number of recent developments in secondary metabolic engineering that could potentially serve as its facilitators. It is hoped that the ever-declining costs of de novo gene synthesis; the improved use of bioinformatic tools to mine, sort and analyze biological data; and the increasing sensitivity and sophistication of investigational tools will make the maturation of microbial metabolic engineering an autocatalytic process. Encouraged by these advances, research groups across the world would take up the challenge of secondary metabolite production in simple hosts with renewed vigor, thereby adding to the range of products synthesized using metabolic engineering.

Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature's favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (∼570 ± 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities.Taxol | P450 | metabolic engineering | natural products | oxygenated taxanes

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