**Jascha Rolf 1, Mattijs K. Julsing 1,2, Katrin Rosenthal <sup>1</sup> and Stephan Lütz 1,\***


Received: 31 March 2020; Accepted: 16 April 2020; Published: 18 April 2020

**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−<sup>1</sup> (corresponding to 3.6 g·L−<sup>1</sup> in the aqueous production phase) were achieved with industrially viable space-time yields of 0.15 g·L−1·h<sup>−</sup>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.

**Keywords:** monoterpenes; limonene; glycerol; mevalonate pathway; reaction engineering; bioprocess; biocatalyst; two-liquid phase fermentation; in situ product removal
