Resting cells of recombinant E. coli show high epoxidation yields on energy source and high sensitivity to product inhibition

Abstract

Metabolically active resting (i.e., nongrowing) bacterial cells have a high potential in cofactor-dependent redox biotransformations. Where growing cells require carbon and energy for biomass production, resting cells can potentially exploit their metabolism more efficiently for redox biocatalysis allowing higher specific activities and product yields on energy source. Here, the potential of resting recombinant E. coli containing the styrene monooxygenase StyAB was investigated for enantioselective styrene epoxidation in a two-liquid phase setup. Resting cells indeed showed twofold higher specific activities as compared to growing cells in a similar setup. However, product formation rates decreased steadily resulting in lower final product concentrations. The low intrinsic stability of the reductase component StyB was found to limit overall biocatalyst stability. Such limitation by enzyme stability was overcome by increasing intracellular StyB levels. Beyond that, product inhibition was identified as a limiting factor, whereas complete toxification of the bacterial cells, as it was observed with growing cells, and deactivation of the multicomponent enzyme system did not occur. The resting cell setup allowed high product yields on glucose of more than 5 mol mol(glucose)(-1), which makes the use of resting cells a promising approach for ecologically as well as economically sustainable oxygenase-based whole-cell biocatalysis.

Comparing in situ removal strategies for improving styrene bioproduction

“…Examples of successful approaches for the in situ recovery of inhibitory biomolecules include solvent extraction [20,23,24], adsorption [25][26][27], gas stripping [28], vacuum stripping [29], and membrane pervaporation [30,31]. Meanwhile, whereas in situ recovery strategies have been demonstrated as effective for improving the bioproduction of other aromatic compounds (e.g., 2-phenylethanol, phydroxystyrene, (S)-styrene oxide, and benzaldehyde) [32][33][34][35][36][37], their application to bio-derived styrene has not yet been reported. Moreover, whereas said prior studies have predominantly focused on single step biotransformations, the alternative focus here on de novo biosynthesis directly from renewable glucose presents new challenges.…”

Proline Availability Regulates Proline-4-Hydroxylase Synthesis and Substrate Uptake in Proline-Hydroxylating Recombinant Escherichia coli

“…Similarly to styrene epoxidation by styrene monooxygenase-containing E. coli (20), oxygenase activities in growing cells were lower than corresponding resting cell activities. In growing cells, the fluxes toward ␣KG were …”

“…4a). Furthermore, while resting cells have a low proline demand for protein synthesis and catabolism, potentially leading to higher proline availability for biocatalysis, TCA cycle activity has been described to be low (20). Along this line of discussion, proline hydroxylation in resting cells appears to be limited by the cosubstrate supply.…”

“…The benefits of metabolic engineering have especially become evident in fermentation processes, as exemplified by the development of strains for the biotechnological production of indigo (8) and propanediol (9). Metabolic engineering can rely on a set of powerful tools, such as flux balance analysis (10) and metabolic flux analysis (MFA) (11-13), used, inter alia, for the optimization of the production of various amino acids in Corynebacterium glutamicum (14,15).The efficiency of whole-cell redox biocatalysts often depends on microbial physiology (16, 17), for example, because the provision of (co)substrates/cofactors becomes limiting (18, 19), or because a toxic or inhibiting product has accumulated (20,21). With respect to cell physiology, 2-oxoacid-dependent nonheme Fe(II)-dependent dioxygenases (22, 23), such as proline-4-hydroxylases (P4Hs), are highly interesting.…”

“…The efficiency of whole-cell redox biocatalysts often depends on microbial physiology (16,17), for example, because the provision of (co)substrates/cofactors becomes limiting (18,19), or because a toxic or inhibiting product has accumulated (20,21). With respect to cell physiology, 2-oxoacid-dependent nonheme Fe(II)-dependent dioxygenases (22,23), such as proline-4-hydroxylases (P4Hs), are highly interesting.…”