Coupling limonene formation and oxyfunctionalization by mixed‐culture resting cell fermentation

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.

Complete Biosynthesis of Anthocyanins Using E. coli Polycultures

“…The study and application of natural microbial consortia have been topics of interest in the scientific literature for decades (6–8); however, the development of synthetic consortia, specifically consortia for metabolic engineering applications, has gained significant traction in the past few years (9–13). Several excellent examples of employing microbial communities for metabolic engineering have resulted in significant improvements over monoculture efforts (14).…”

Decoupling production from growth by magnesium sulfate limitation boosts de novo limonene production

“…Typically, the production of terpenes, such as limonene (Alonso‐Gutierrez et al, ; Willrodt et al, ), pinene (Sarria et al, ), or sclareol (Schalk et al, ) was based on growing Escherichia coli cells, co‐producing the terpene alongside major quantities of by‐products (e.g., biomass). Resting cells only have been applied for a mixed strain approach to produce perillyl acetate (Willrodt et al, ).…”

Biocatalytic activity of Monascus mycelia depending on physiology and high sensitivity to product concentration

“…Accumulation of high concentration of extracellular Monascus pigments was realized due to the plant oil phase with high extractive capacity. Furthermore, instable/toxic extracellular product can also be converted into relatively stable/nontoxic compound by enzymatic reaction (Willrodt et al 2015) or non-enzymatic reaction (Xiong et al 2015; Domaille et al 2016; Wallace and Balskus 2016), which may be a more efficient strategy to make whole cell biocatalyst play its potential.…”

“…In-situ product removal, such as addition of solid-state adsorbent (Evanst and Wang 1984), nonaqueous two-phase extraction (Wang and Dai 2010), is the most common strategy for elimination of product inhibition/degradation. Cascade conversion of instable/toxic product into stable/non-toxic compound by enzymatic reaction (Willrodt et al 2015) or non-enzymatic reaction (Xiong et al 2015; Domaille et al 2016; Wallace and Balskus 2016) is also developed for bioprocess optimization.…”