The future of metabolic engineering and synthetic biology: Towards a systematic practice

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.National Institutes of Health (U.S.) (1-R01-GM085323-01A1)Special Research Funds BOF (BOF08/PDO/014)Research Foundation Flanders (FWO-Vlaandern V.4.174.10.N.01

Metabolic engineering for high-level flavonoids and stilbenes production in microorganisms

This dissertation represented an effort in efficient biosynthesis of both natural and unnatural stilbenes and flavonoids in microbial platform utilizing state of the art metabolic engineering strategies. Flavonoids are plant secondary metabolites that possess a variety of nutraceutical and pharmaceutical properties, also the potential use as natural colorants. Therefore, it was of interest to reconstitute the entire flavonoids biosynthetic pathway in heterologous host, specifically microorganisms such as E. coli and S. cerevasiae due to their versatility and short doubling time. The present work focused on the biosynthesis of three high-valued compounds: stilbenes and two flavonoids, namely isoflavonoids and anthocyanins. First, their specific pathways were established in microorganisms. Next, different metabolic engineering strategies were utilized to optimize the production of individual molecule: (i) Identify catalytic protein with highest activity or best substrate specificity from a variety of plant homologs; (ii) Optimize the "construct environment" through promoter, vector, host strain...; (iii) Pinpoint the rate limiting precursor and identify possible metabolic intervention targets; (iv) Improve culture conditions: carbon source selection, inducer concentration, induction time and medium. The optimized platform allowed downstream assessment of their medicinal properties, such as the inhibition against digestive enzyme, α-glucosidase and binding affinity towards human estrogen receptor α and β

Engineering terpenoid natural product biosynthesis in Escherichia coli

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Chemistry & Biology Article A Versatile Microbial System for Biosynthesis of Novel Polyphenols with Altered Estrogen Receptor Binding Activity

SUMMARY Isoflavonoids possess enormous potential for human health with potential impact on heart disease and cancer, and some display striking affinities for steroid receptors. Synthesized primarily by legumes, isoflavonoids are present in low and variable abundance within complex mixtures, complicating efforts to assess their clinical potential. To satisfy the need for controlled, efficient, and flexible biosynthesis of isoflavonoids, a three-enzyme system has been constructed in yeast that can convert natural and synthetic flavanones into their corresponding isoflavones in practical quantities. Based on the determination of the substrate requirements of isoflavone synthase, a series of natural and nonnatural isoflavones were prepared and their binding affinities for the human estrogen receptors (ERa and ERb) were determined. Structure activity relationships are suggested based on changes to binding affinities related to small variations on the isoflavone structure

Efficient utilization of pentoses for bioproduction of the renewable two-carbon compounds ethylene glycol and glycolate

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