Publication Details

Category Text Publication
Reference Category Journals
DOI 10.1002/bit.25592
Title (Primary) Coupling limonene formation and oxyfunctionalization by mixed-culture resting cell fermentation
Author Willrodt, C.; Hoschek, A.; Bühler, B.; Schmid, A.; Julsing, M.K.
Source Titel Biotechnology and Bioengineering
Year 2015
Department SOMA
Volume 112
Issue 9
Page From 1738
Page To 1750
Language englisch
Keywords industrial biotechnology; fermentation; oxygenated monoterpenoids; limonene; cytochrome P450; metabolic and reaction engineering
UFZ wide themes MIKAT; RU4;
Abstract Metabolic engineering strategies mark a milestone for the fermentative production of bulk and fine chemicals. Yet, toxic products and volatile reaction intermediates with low solubilities remain challenging. Prominent examples are artificial multistep pathways like the production of perillyl acetate (POHAc) from glucose via limonene. For POHAc, these limitations can be overcome by mixed-culture fermentations. A limonene biosynthesis pathway and cytochrome P450 153A6 (CYP153A6) as regioselective hydroxylase are used in two distinct recombinant E. coli. POHAc formation from glucose in one recombinant cell was hindered by ineffective coupling of limonene synthesis and low rates of oxyfunctionalization. The optimization of P450 gene expression led to the formation of 6.20 ± 0.06 mg gcdw−1 POHAc in a biphasic batch cultivation with glucose as sole carbon and energy source. Increasing the spatial proximity between limonene synthase and CYP153A6 by a genetic fusion of both enzymes changed the molar limonene/POHAc ratio from 3.2 to 1.6. Spatial separation of limonene biosynthesis from its oxyfunctionalization improved POHAc concentration 3.3-fold to 21.7 mg L−1 as compared to a biphasic fermentation. Mixed-cultures of E. coli BL21 (DE3) containing the limonene biosynthesis pathway and E. coli MG1655 harboring either CYP153A6, or alternatively a cymene monooxygenase, showed POHAc formation rates of 0.06 or 0.11 U gcdw−1, respectively. This concept provides a novel framework for fermentative syntheses involving toxic, volatile, or barely soluble compounds or pathway intermediates
Persistent UFZ Identifier
Willrodt, C., Hoschek, A., Bühler, B., Schmid, A., Julsing, M.K. (2015):
Coupling limonene formation and oxyfunctionalization by mixed-culture resting cell fermentation
Biotechnol. Bioeng. 112 (9), 1738 - 1750 10.1002/bit.25592