A Gram-Scale Limonene Production Process with Engineered Escherichia coli.

Jascha Rolf,Stephan Lütz,Mattijs K Julsing,Katrin Rosenthal

doi:10.3390/molecules25081881

Jascha Rolf, Stephan Lütz + Show 2 more

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https://doi.org/10.3390/molecules25081881

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Abstract

Monoterpenes, such as the cyclic terpene limonene, are valuable and important natural products widely used in food, cosmetics, household chemicals, and pharmaceutical applications. The biotechnological production of limonene with microorganisms may complement traditional plant extraction methods. For this purpose, the bioprocess needs to be stable and ought to show high titers and space-time yields. In this study, a limonene production process was developed with metabolically engineered Escherichia coli at the bioreactor scale. Therefore, fed-batch fermentations in minimal medium and in the presence of a non-toxic organic phase were carried out with E. coli BL21 (DE3) pJBEI-6410 harboring the optimized genes for the mevalonate pathway and the limonene synthase from Mentha spicata on a single plasmid. The feasibility of glycerol as the sole carbon source for cell growth and limonene synthesis was examined, and it was applied in an optimized fermentation setup. Titers on a gram-scale of up to 7.3 g·Lorg−1 (corresponding to 3.6 g·L−1 in the aqueous production phase) were achieved with industrially viable space-time yields of 0.15 g·L−1·h−1. These are the highest monoterpene concentrations obtained with a microorganism to date, and these findings provide the basis for the development of an economic and industrially relevant bioprocess.

Highlights

Monoterpenes are volatile, lipophilic compounds in the essential oils of plants, which often find application as flavors and fragrances in food, cosmetics, and household chemicals
This study aims at the development of a feasible bioreactor scale process for monoterpene production with a recombinant E. coli strain that is genetically optimized for limonene synthesis
The use of glycerol as a carbon source resulted in higher limonene formation rates, a prolonged growth phase, and increased stability compared to the same whole-cell biocatalyst growing on glucose [13]

Summary

Introduction

Monoterpenes are volatile, lipophilic compounds in the essential oils of plants, which often find application as flavors and fragrances in food, cosmetics, and household chemicals. Limonene is the predominant monoterpene in the essential oils of citrus fruits and can be found in oaks, pines, and spearmint as well. Limonene shows antimicrobial properties [5], can be functionalized because of its two double bonds [6], and finds application as a building block for several commodity chemicals and pharmaceuticals. The oxygenated derivatives of limonene show potent pharmaceutical activities. Perillyl alcohol, which can be obtained by the regiospecific oxygenation of limonene via whole-cell biotransformation [7,8], has proven anti-cancer properties [9]. The application of monoterpenes as starting materials for industrially or pharmaceutically relevant compounds requires efficient synthesis routes [10].

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