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

Abstract

Microbial physiology plays a crucial role in whole-cell biotransformation, especially for redox reactions that depend on carbon and energy metabolism. In this study, regio-and enantio-selective proline hydroxylation with recombinant Escherichia coli expressing proline-4-hydroxylase (P4H) was investigated with respect to its interconnectivity to microbial physiology and metabolism. P4H production was found to depend on extracellular proline availability and on codon usage. Medium supplementation with proline did not alter p4h mRNA levels, indicating that P4H production depends on the availability of charged prolyl-tRNAs. Increasing the intracellular levels of soluble P4H did not result in an increase in resting cell activities above a certain threshold (depending on growth and assay temperature). Activities up to 5-fold higher were reached with permeabilized cells, confirming that host physiology and not the intracellular level of active P4H determines the achievable whole-cell proline hydroxylation activity. Metabolic flux analysis revealed that tricarboxylic acid cycle fluxes in growing biocatalytically active cells were significantly higher than proline hydroxylation rates. Remarkably, a catalysis-induced reduction of substrate uptake was observed, which correlated with reduced transcription of putA and putP, encoding proline dehydrogenase and the major proline transporter, respectively. These results provide evidence for a strong interference of catalytic activity with the regulation of proline uptake and metabolism. In terms of whole-cell biocatalyst efficiency, proline uptake and competition of P4H with proline catabolism are considered the most critical factors. I n redox biocatalysis, especially when activation of molecular oxygen is involved, the whole cell still remains the preferred catalyst. Besides the capability to continuously regenerate redox cofactors via central carbon metabolism, the whole-cell approach provides advantages with respect to operational catalyst stability, e.g., via continuous (multicomponent) enzyme synthesis, assembly, and stabilization, as well as degradation of reactive oxygen species (1-3).Oxygenases are of special interest for industrial applications, since they catalyze the highly specific oxyfunctionalization of unactivated C-H bonds under mild conditions, with molecular oxygen as the oxygen donor (4). The efficiency of an oxygenase-containing whole-cell biocatalyst depends on both enzyme and host cell performance. Hence, biocatalyst engineering must encompass both factors and be integrated with biochemical process engineering (5, 6). The technological progress in enzyme engineering of the recent decades now allows fast generation of enzyme variants with improved properties (7). For in vivo applications, intracellular conditions have to be considered, and host cell engineering often is necessary to allow the exploitation of the catalytic capability of (engineered) enzymes. The benefits of metabolic engineering have especially become evident in fermentation processes, as ex...

Efficient production oftrans-4-Hydroxy-l-proline from glucose by metabolic engineering of recombinantEscherichia coli

“…a), which comprises (i) degradation of l ‐proline caused by proline dehydrogenase (PutA) (Falcioni et al . ), (ii) biosynthesis of succinate from 2‐OG due to the existence of α ‐ketoglutarate dehydrogenase (SucAB), (iii) glyoxylate pathway from isocitrate to succinate affected by isocitrate lyase (AceA), and (iv) isocitrate dehydrogenase (IDH) phosphorylation under the action of isocitrate dehydrogenase kinase/phosphatase (AceK) (Smirnov et al . ).…”

An artificial TCA cycle selects for efficient α‐ketoglutarate dependent hydroxylase catalysis in engineered Escherichia coli

“…The fact that the yield of acetate on glucose was 20% lower at similar growth rate and final biomass titer indicates carbon redistribution in favor of hyp formation when PutP is overproduced. Increasing the substrate concentration (from 5 to 50 mM) did not enhance the hyp formation rate of the 3Δ sucA (pEB_p4h1of_putP) strain, indicating that, already at 5 mM, both the transporter and the P4H work at V max , as can be concluded from their K m values for proline of 3.3 μM and 1 mM, respectively (Falcioni et al, ) (Table ; Supplemental Fig. S7).…”

Biosynthesis of trans-4-hydroxyproline by recombinant strains of Corynebacterium glutamicum and Escherichia coli

“…The gene of trans -P4H from Dactylosporangium sp. ( p4hD ) had been expressed in E. coli successfully and can transform L-proline with good enzymatic properties [11-13,24-26]. The length of p4hD was 816 bps encoding a 272-amino-acid polypeptide with the molecular weight of 29,715 daltons [11,26].…”