Indigo Formation and Rapid NADPH Consumption Provide Robust Prediction of Raspberry Ketone Synthesis by Engineered Cytochrome P450 BM3

Abstract

Natural raspberry ketone has a high value in the flavor, fragrance and pharmaceutical industries. Its extraction is costly, justifying the search for biosynthetic routes. We hypothesized that cytochrome P450 BM3 (P450 BM3) could be engineered to catalyze the hydroxylation of 4-phenyl-2-butanone, a naturally sourceable precursor, to raspberry ketone. The synthesis of indigo by variants of P450 BM3 has previously served as a predictor of promiscuous oxidation reactions. To this end, we screened 53 active-site variants of P450 BM3 using orthogonal high-throughput workflows to identify the most streamlined route to all indigo-forming variants. Among the three known and 13 new indigo-forming variants, eight hydroxylated 4-phenyl-2-butanone to raspberry ketone. Previously unreported variant A82Q displayed the highest initial rates and coupling efficiencies in synthesis of indigo and of raspberry ketone. It produced the highest total concentration of raspberry ketone despite producing less total indigo than previously reported variants. Its productivity, although modest, clearly demonstrates the potential for development of a biocatalytic route to raspberry ketone. In addition to validating indigo as a robust predictor of this promiscuous activity, we demonstrate that monitoring rapid NADPH consumption serves as an alternative predictor of a promiscuous reactivity in P450 BM3.

Design and engineering of neuroglobin to catalyze the synthesis of indigo and derivatives for textile dyeing

“…This activity is almost 2-fold that of the double mutant A15C/H64D Ngb (k cat = 3.89 ± 0.07 min −1 , K m = 0.151 ± 0.008 mM, and k cat /K m = 29 M −1 s −1 ), exceeding that of the recently reported CYP BM3 F87A mutant utilizing H 2 O 2 as a terminal oxidant (k cat = 3.51 ± 0.28 min −1 , K m = 0.13 ± 0.01 mM, and k cat /K m = 450 M −1 s −1 ). 32 Note that CYP BM3 mutants showed much higher catalytic efficiency when supplemented with O 2 and the expensive cofactor NAD(P)H. 33 The catalytic efficiency of Ngb mutants was also compared with those of Mb mutants (Fig. 4B), and the activity of the double and triple mutants is almost 2-fold and 4-fold higher than that of the most efficient mutant, F43Y/H64D Mb, 35 respectively.…”

“…As reviewed recently by Fabara and Fraaije, 23 a large number of metalloenzymes have been exploited for the biosynthesis of indigo and derivative dyes, including naphthalene/biphenyl dioxygenases, 24,25 flavincontaining monooxygenase, [26][27][28] and heme-containing cytochromes P450 (CYPs). [29][30][31][32][33] Meanwhile, in vivo biosynthesis of indigo results in difficult isolation of the product from the bacterial cultures, whereas an in vitro enzymatic reaction may require the addition of expensive NAD(P)H cofactors, [29][30][31][32][33] although unspecific peroxygenases require only H 2 O 2 . 23 To mimic the heme enzyme, Rebelo and co-workers used a reaction system of Fe-porphyrin/H 2 O 2 to catalyze the oxidation of indole in ethanol, producing a low yield of indigo (20%).…”

The beauty of biocatalysis: sustainable synthesis of ingredients in cosmetics

“…para -hydroxylation of the phenyl ring) with one variant, A82Q, showing increased rate of production of raspberry ketone. 354…”

Growth-Based, High-Throughput Selection for NADH Preference in an Oxygen-Dependent Biocatalyst

“…High-throughput screening methods are essential to the enzyme engineering workflow. Compared to other methods using 96 well plates, , microfluidics, , and fluorescence sorting based assays, cell growth-based selections are more facile and scalable because they utilize cell survival as an easy readout. Furthermore, the selected enzyme variants are directly compatible with in vivo applications.…”