Discovery of Novel Bacterial Chalcone Isomerases by a Sequence‐Structure‐Function‐Evolution Strategy for Enzymatic Synthesis of (S)‐Flavanones

Abstract Chalcone isomerase (CHI) is a key enzyme in the biosynthesis of flavonoids in plants. The first bacterial CHI (CHIera) was identified from Eubacterium ramulus, but its distribution, evolutionary source, substrate scope, and stereoselectivity are still unclear. Here, we describe the identification of 66 novel bacterial CHIs from Genbank using a novel Sequence‐Structure‐Function‐Evolution (SSFE) strategy. These novel bacterial CHIs show diversity in substrate specificity towards various hydroxylated and methoxylated chalcones. The mutagenesis of CHIera according to the substrate binding models of these novel bacterial CHIs resulted in several variants with greatly improved activity towards these chalcones. Furthermore, the preparative scale conversion catalyzed by bacterial CHIs has been performed for five chalcones and revealed (S)‐selectivity with up to 96 % ee, which provides an alternative biocatalytic route for the synthesis of (S)‐flavanones in high yields.


NMR experiments
The quantification of the products was carried out using a Bruker Ascend 600 (B0 = 14.1 T, Bruker Biospin, Rheinstetten, Germany) with resonance frequencies of 600 MHz for 1 H and 151 MHz for 13 C, respectively. The solvent DMSO-d6 (δ( 1 H) = 2.50 ppm, δ( 13 C) = 39.52 ppm) was used for chemical shifts referencing for all spectra. The samples contained traces of n-hexane, ethyl acetate and acetone which are denoted in the corresponding spectra. qNMR: Three samples were prepared for each sample material using an amount of ~2-7 mg sample and quantification reference, respectively. To also minimize errors due to the integration procedure and the bias of the spectrometer all samples were measured three times and analyzed. 1,2,4,5-Tetrachloro-3-nitrobenzene (TraceCERT ® ) with a purity of 99,82% was used as quantification reference (QR) in all analysis. For each of the flavanones (1b-5b) the area of peak (IAnalyte) of the CH group at position 2 at ~5.4 ppm was used and correlated with the area peak of the QR (IQR) under considering the molar masses of the analyte (MAnalyte) and the QR (MQR) as well as the purity of the QR (PQR). The formula is denoted below and was provided with the TraceCERT ® certificate of the QR. For the chalcone 5a (4-O-methylbutein) the signals of 2 protons around 7.7 ppm were used as the analyte integral (IAnalyte). According to the sample structure the number of nuclei per molecule represented by the integrated signals was NAnalyte = NQR = 1 for the flavanones (5.4 ppm) and NQR = 2 for the chalcone 4-Omethylbutein (5a).

Enzymatic synthesis of naringenin (1b) by CHIera
The enzymatic reactions were carried out by dropping 35 mg of naringenin chalcone (in 1 mL MeOH stock) into 100 mL PBS (50 mM, pH 8.0) containing 2 mg of CHIera. The reaction was stopped when there was no color change in the reaction solution (~10 min). The product was extracted by 200 mL ethyl acetate directly after the reaction and dried by magnesium sulfate anhydrous. The solvent and water were removed by using a rotary evaporator. The product (41.9 mg yellow powder) was obtained after lyophilization for 2 days. The chemical structure and purity (88.2 ± 7.1%) of the product was determined by NMR as descripted above. The yield was ~100%.

Enzymatic synthesis of eriodictyol (2b) by CHIeraMut5
The enzymatic reactions were carried out by dropping 30 mg of eriodictyol chalcone (in 1 mL MeOH stock) into 100 mL PBS (50 mM, pH 8.0) containing 2.7 mg of CHIeraMut5. The reaction was stopped by adding 200 mL MeOH when there was no color change in the reaction solution (~10 min). The reaction solution was filtered to remove the precipitate. The solvent and water were removed by using a rotary evaporator. The product (95.5 mg yellow powder) was obtained after lyophilization for 2 days. The chemical structure and purity (37.8 ± 3.5%) of the product was determined by NMR as descripted above. The yield was ~100%. 1

Enzymatic synthesis of homoeriodictyol (3b) by CHIeraMut5
The enzymatic reactions were carried out by dropping 30 mg of homoeriodictyol chalcone (in 1 mL DMSO stock) into 100 mL PBS (50 mM, pH 8.0) containing 3 mg of CHIeraMut5. The reaction was stopped when there was no color change in the reaction solution (~10 min). The product was extracted by 200 mL ethyl acetate directly after the reaction and dried by magnesium sulfate anhydrous. The solvent and water were removed by using a rotary evaporator. The product (48.5 mg yellow powder) was obtained after lyophilization for 2 days. The chemical structure and purity (63.4 ± 5.2%) of the product was determined by NMR as descripted above.

Enzymatic synthesis of 7,3'-dihydroxy-4'-methoxyflavanone (5b) by CHIeraMut11
The enzymatic reactions were carried out by dropping 30 mg of 4-O-methylbutein (in 1 mL DMSO stock) into 100 mL PBS (50 mM, pH 8.0) containing 16.8 mg of CHIeraMut11. The reaction was stopped after 2 days. The product was extracted by 200 mL ethyl acetate directly after the reaction and dried by magnesium sulfate anhydrous. The solvent and water were removed by using a rotary evaporator. The product (52.3 mg yellow powder) was obtained after lyophilization for 2 days. The chemical structure and purity (48.9 ± 3.8%) of the product was determined by NMR as descripted above. The yield was 85%.

Branch Access no. A-ring C-ring
The proteins selected for activity determination are highlighted in red. "-" means gap in the protein sequence alignment.