Combining Genotype Improvement and Statistical Media Optimization for Isoprenoid Production in E. coli

Kang Zhou,Arnold Driessen,Congqiang Zhang,Xixian Chen,Gregory Stephanopoulos,Heng-Phon Too,Ruiyang Zou

doi:10.1371/journal.pone.0075164

Abstract

Isoprenoids are a large and diverse class of compounds that includes many high value natural products and are thus in great demand. To meet the increasing demand for isoprenoid compounds, metabolic engineering of microbes has been used to produce isoprenoids in an economical and sustainable manner. To achieve high isoprenoid yields using this technology, the availability of metabolic precursors feeding the deoxyxylulose phosphate (DXP) pathway, responsible for isoprenoid biosynthesis, has to be optimized. In this study, phosphoenolpyruvate, a vital DXP pathway precursor, was enriched by deleting the genes encoding the carbohydrate phosphotransferase system (PTS) in E. coli. Production of lycopene (a C40 isoprenoid) was maximized by optimizing growth medium and culture conditions. In optimized conditions, the lycopene yield from PTS mutant was seven fold higher than that obtained from the wild type strain. This resulted in the highest reported specific yield of lycopene produced from the DXP pathway in E. coli to date (20,000 µg/g dry cell weight). Both the copy number of the plasmid encoding the lycopene biosynthetic genes and the expression were found to be increased in the optimized media. Deletion of PTS together with a similar optimization strategy was also successful in enhancing the production of amorpha-1,4-diene, a distinct C15 isoprenoid, suggesting that the approaches developed herein can be generally applied to optimize production of other isoprenoids.

Highlights

Isoprenoids constitute one of the most diverse classes of secondary metabolites in nature, with more than 55,000 distinct compounds [1]
As PEP is known to be consumed by the phosphotransferase system (PTS) when carbohydrates are imported [6], it is worth investigating whether the deletion of PTS would increase intracellular PEP concentration which may result in the enhancement of isoprenoid production
PEP can be converted to phosphoglycerates and to glyceraldehyde 3-phosphate (GAP), the limiting precursor of the D-xylulose 5-phosphate (DXP) pathway (Figure 1) [20]

Summary

Introduction

Isoprenoids constitute one of the most diverse classes of secondary metabolites in nature, with more than 55,000 distinct compounds [1]. An approach where central metabolism was genetically rerouted to increase PEP concentration was shown to be effective in enhancing the production of lycopene, an important isoprenoid compound [5]. As PEP is known to be consumed by the phosphotransferase system (PTS) when carbohydrates are imported [6], it is worth investigating whether the deletion of PTS would increase intracellular PEP concentration which may result in the enhancement of isoprenoid production. Deletion of PTS caused severe growth retardation when E. coli was grown in media containing glucose as the sole carbon source. This resulted in low cell density and a lower total yield of lycopene from the PTS knockout E. coli

Methods

Results

Discussion

Conclusion