Regioselective Hydroxylation of Stilbenes by Engineered Cytochrome P450 from Thermobifida fusca YX

Abstract

Since the past decades, the plant stilbenoid resveratrol has gained significant attention of the general public as well as the research community due to its versatile medicinal properties. Apart from resveratrol, there is also an increasing interest in other plant stilbenoids because of their different potential biological activities. In order to meet the increasing demand for stilbenoids, alternative and sustainable approaches for their production are needed. We identified the cytochrome P450 monooxygenase 154E1 from Thermobifida fusca YX (CYP154E1) which enables the synthesis of (E)‐4,4′‐dihydroxystilbene via direct double hydroxylation of (E)‐stilbene. The construction of a triple mutant led to a more than six‐fold increased catalytic efficiency compared to the wild type enzyme. CYP154E1 and variants thereof accepted not only (E)‐stilbene but also possessed remarkable activity towards ortho‐ and meta‐substituted hydroxystilbenes leading to resveratrol, (E)‐2,4′‐dihydroxystilbene, (E)‐2,4′,5‐trihydroxystilbene and (E)‐3,4′‐dihydroxystilbene. The combination of protein engineering and the use of methyl‐β‐cyclodextrin as substrate solubilizing agent resulted in product titers of up to 4.2 g L−1 and enzyme total turnover numbers (TTN) of up to 20,000.magnified image

Selective synthesis of the resveratrol analogue 4,4′-dihydroxy-trans-stilbene and stilbenoids modification by fungal peroxygenases

“…32 The enzyme showed a similar parahydroxylating regioselectivity for trans-stilbene and similar conversion rates as reported herein for the UPOs but with considerably lower TTN (4800) as compared with AaeUPO (200 000) and MroUPO (25 000). Comparing kinetic data, CYP154E1 and its variants exhibited lower affinities (higher K m values) and turnover numbers, and hence a thousand-times lower catalytic efficiency compared to AaeUPO.…”

Characterization of CYP154F1 from Thermobifida fusca YX and Extension of Its Substrate Spectrum by Site‐Directed Mutagenesis

“…The space‐creating mutations T234V and T234A enabled oxidation of the relatively bulky molecule ( E )‐stilbene. Similar to CYP154F1, we reported an increase in activity towards ( E )‐stilbene by changing isoleucine at the homologous position 238 in CYP154E1 to alanine or valine (I238V/A) …”

“…Replacement of alanine to glycine at position 235 seems to be co-responsible for this effect because this single mutation also led to atotal loss of CYP154F1 activity.Remarkably,i nC YP154E1, the replacement of glycineb ya lanine at the corresponding position (G239A) led to as ixfoldi ncrease in activity towards (E)-stilbene. [12] The equivalent substitution, G248A, in P450cam from P. putida improved oxidation activity and coupling efficiency towards ethane. [21] This positioni sl ocated in the center of the Ih elix and belongs to substrate recognition site 4( SRS4), [22] and is usually occupied by glycine or alanine.…”

“…Out of 51 organic compounds of different size and chemical structure, 33 were oxidized by CYP154E1 and 23 by CYP154A8 . Recently, we reported a high activity and promising biotechnological application of CYP154E1 for the synthesis of various stilbenoids . Thus, the characterized CYP154 enzymes belonging to the same family accept substances from various chemical groups.…”

“…of various stilbenoids. [12] Thus, the characterized CYP154 enzymes belonging to the same family accept substances from variousc hemical groups. Such substrate promiscuity makes these enzymes clear targetsfor biocatalysis.…”