Engineering the productivity of recombinant Escherichia coli for limonene formation from glycerol in minimal media

Abstract

The efficiency and productivity of cellular biocatalysts play a key role in the industrial synthesis of fine and bulk chemicals. This study focuses on optimizing the synthesis of (S)-limonene from glycerol and glucose as carbon sources using recombinant Escherichia coli. The cyclic monoterpene limonene is extensively used in the fragrance, food, and cosmetic industries. Recently, limonene also gained interest as alternative jet fuel of biological origin. Key parameters that limit the (S)-limonene yield, related to genetics, physiology, and reaction engineering, were identified. The growth-dependent production of (S)-limonene was shown for the first time in minimal media. E. coli BL21 (DE3) was chosen as the preferred host strain, as it showed low acetate formation, fast growth, and high productivity. A two-liquid phase fed-batch fermentation with glucose as the sole carbon and energy source resulted in the formation of 700 mg L(org) (-1) (S)-limonene. Specific activities of 75 mU g(cdw) (-1) were reached, but decreased relatively quickly. The use of glycerol as a carbon source resulted in a prolonged growth and production phase (specific activities of ≥50 mU g(cdw) (-1) ) leading to a final (S)-limonene concentration of 2,700 mg L(org) (-1) . Although geranyl diphosphate (GPP) synthase had a low solubility, its availability appeared not to limit (S)-limonene formation in vivo under the conditions investigated. GPP rerouting towards endogenous farnesyl diphosphate (FPP) formation also did not limit (S)-limonene production. The two-liquid phase fed-batch setup led to the highest monoterpene concentration obtained with a recombinant microbial biocatalyst to date.

Functional characterization of a geraniol synthase-encoding gene from Camptotheca acuminata and its application in production of geraniol in Escherichia coli

“…The present work showed that geraniol production was increased to 48.5 ± 0.9 mg L −1 by engineering tCaGES and the foreign genes mentioned above. The geraniol yield of this study is higher than those in transgenic tobacco and yeast; also, parallel to or higher than the yields of other terpenes production in metabolically engineered microorganisms [23,32,39,40,42]. Recently it has been demonstrated that deletion of YjgB, an endogenous dehydrogenase responsible for geraniol loss in E. coli MG1655, will increase geraniol production to 182.5 mg L −1 [38].…”

“…In addition, the fed-batch fermentation was proved to increase the yields of other terpenes [23,39,40,42]. Thus, this study will be an alternative to selectively produce geraniol in engineered E. coli, together with other metabolic engineering methods such as balancing the whole biosynthetic pathway and using fed-batch fermentation.…”

Microbial production of scent and flavor compounds

“…Limonene fermentation using glycerol as a carbon source has also been explored [44 ]. Glycerol provides a prolonged growth and production phase, achieving titers as high as 2.7 g/L (Table 1) [44 ].…”

“…Glycerol provides a prolonged growth and production phase, achieving titers as high as 2.7 g/L (Table 1) [44 ].…”

Bioprocess Engineering for Microbial Synthesis and Conversion of Isoprenoids

“…Considering the isoprenoid bioprocesses with direct contact and internal removal listed in Table 1 it is notable that only laboratory-scale systems with total reaction volumes typically below 10 L have been published and that most publications describe a proof-of-concept without further investigating product purification from the receiving phase. Exceptions are the distillation of limonene produced by recombinant E. coli from diisononyl phthalate [107], the purification of artemisinic acid from isopropyl myristate [65], and a stirred-tank reactor operated in continuous cultivation mode with a continuous recovery of the product α-santalene and regeneration of the extractive solvent dodecane [78]. On the shaking flask scale the recovery of bisabolene from the organic phase and its purification and subsequent chemical treatment have been described [67].…”