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Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases

Paper ID Volume ID Publish Year Pages File Format Full-Text
31618 44824 2011 10 PDF Available
Title
Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases
Abstract

Expression of foreign pathways often results in suboptimal performance due to unintended factors such as introduction of toxic metabolites, cofactor imbalances or poor expression of pathway components. In this study we report a 120% improvement in the production of the isoprenoid-derived sesquiterpene, amorphadiene, produced by an engineered strain of Escherichia coli developed to express the native seven-gene mevalonate pathway from Saccharomyces cerevisiae ( Martin et al. 2003). This substantial improvement was made by varying only a single component of the pathway (HMG-CoA reductase) and subsequent host optimization to improve cofactor availability. We characterized and tested five variant HMG-CoA reductases obtained from publicly available genome databases with differing kinetic properties and cofactor requirements. The results of our in vitro and in vivo analyses of these enzymes implicate substrate inhibition of mevalonate kinase as an important factor in optimization of the engineered mevalonate pathway. Consequently, the NADH-dependent HMG-CoA reductase from Delftia acidovorans, which appeared to have the optimal kinetic parameters to balance HMG-CoA levels below the cellular toxicity threshold of E. coli and those of mevalonate below inhibitory concentrations for mevalonate kinase, was identified as the best producer for amorphadiene (54% improvement over the native pathway enzyme, resulting in 2.5 mM or 520 mg/L of amorphadiene after 48 h). We further enhanced performance of the strain bearing the D. acidovorans HMG-CoA reductase by increasing the intracellular levels of its preferred cofactor (NADH) using a NAD+-dependent formate dehydrogenase from Candida boidinii, along with formate supplementation. This resulted in an overall improvement of the system by 120% resulting in 3.5 mM or 700 mg/L amorphadiene after 48 h of fermentation. This comprehensive study incorporated analysis of several key parameters for metabolic design such as in vitro and in vivo kinetic performance of variant enzymes, intracellular levels of protein expression, in-pathway substrate inhibition and cofactor management to enable the observed improvements. These metrics may be applied to a broad range of heterologous pathways for improving the production of biologically derived compounds.

► Variant HMG-CoA reductases to optimize mevalonate pathway engineered in E. coli. ► Differences in cofactor specificities and kinetic properties. ► Key factors for optimization—HMG-CoA toxicity and mevalonate kinase inhibition. ► NADH-dependent HMG-CoA reductase from Delftia acidovorans is best performer. ► Further improvement achieved by enhancing intracellular NADH levels.

Keywords
Mevalonate pathway; Metabolic pathway optimization; HMG-CoA Reductase; E. coli; Cofactor regeneration; Mevalonate kinase
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Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 13, Issue 5, September 2011, Pages 588–597
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us