Highly E ﬀ ective, Regiospeciﬁc Hydrogenation of Methoxychalcone by Yarrowia lipolytica Enables Production of Food Sweeteners

: We describe the impact of the number and location of methoxy groups in the structure of chalcones on the speed and e ﬃ ciency of their transformation by unconventional yeast strains. The e ﬀ ect of substrate concentration on the conversion e ﬃ ciency in the culture of the Yarrowia lipolytica KCh 71 strain was tested. In the culture of this strain, monomethoxychalcones (2 (cid:48) -hydroxy-2 (cid:48)(cid:48) -, 3 (cid:48)(cid:48) and 4 (cid:48)(cid:48) -methoxychalcone) were e ﬀ ectively hydrogenated at over 40% to the speciﬁc dihydrochalcones at a concentration of 0.5 g / L of medium after just 1 h of incubation. A conversion rate of over 40% was also observed for concentrations of these compounds of 1 g / L of medium after three hours of transformation. As the number of methoxy substituents increases in the chalcone substrate, the rate and e ﬃ ciency of transformation to dihydrochalcones decreased. The only exception was 2 (cid:48) -hydroxy-2 (cid:48)(cid:48) ,5 (cid:48)(cid:48) -dimethoxychalcone, which was transformed into dihydrochalcone by strain KCh71 with a yield comparable to that of chalcone containing a single methoxy group.


Introduction
Sweet taste plays a dominant role in human food preferences [1]. It is the most important sensory feature of food products. It is not only a source of pleasure but also a basic energy stimulus for the body. Prospective studies have provided information on the correlation between excessive consumption of sugar and sugar-rich products, and an increased risk of pancreatic cancer. Given the role of hyperglycaemia and hyperinsulinaemia in the development of this cancer, it has been established that the direct cause of organ tissue death is the induction of frequent food hyperglycaemia and the increase in demand and reduction of insulin sensitivity [2]. There is also growing evidence of the role of increased sugar consumption in the development of hypertension, inflammation, and coronary artery disease [3][4][5].
The results of each reaction were analysed in two aspects: (a) strains performing effective hydrogenation of as many substrates as possible, regardless of their structure. Considering this criterion, the best biocatalyst turned out to be the Yarrowia lipolytica KCh 71 strain ( Table 2). Similar efficiencies of this strain have also been described during biotransformation of chalcone having heteroatom in the B ring [23]; (b) most efficiently converted substrate. In this case, with the increasing number of methoxyl groups in the B ring, the conversion efficiency of the substrate decreased, while in the presence of methoxyl substituents in the A ring, the product did not occur at all. These results were noted for most of the tested biocatalysts ( Also, the methoxyl substitution position affected the rate and efficiency of the hydrogenation process. These differences are seen the best in the R. glutinis KCh 242 strain, in a culture whose conversion of compounds containing one methoxyl group in various positions (in the same ring) is diametrically different. Substrate 1 having a methoxyl group at the C-2" carbon was 14% converted after seven days of incubation, whereas for substrates 2 and 3 (methoxyl group located at C-3" and C-4", respectively) conversion rates exceeded 90% after seven days ( Figure 1A). Another preference, selectivity due to the structure of the substrate, was observed for the strain Saccharomyces cerevisiae KCh 464, for which a significantly higher conversion was recorded for compound 3. Compounds with the methoxy group located at the C-2"and C-3" carbon demonstrate a much slower conversion in the culture of this strain (Figure 1 B). This observation was surprising because strains of this species in many papers are described as effective and universal biocatalysts for the hydrogenation of double bonds, both in chalcones [13,20] and other compounds containing the enone moiety [45,46].  3 ± 0.6 4 ± 0.6 5 ± 0.6 6 ± 1.5 7 ± 1.5 Based on screening tests, we decided to choose Yarrowia lipolytica KCh 71 strain for further experiments. This strain showed the highest conversion and the largest number of substrates tested after just one day of biotransformation. In addition, yeast from this species was authorised in 2019 as a novel food (EU Regulation 2019/760). The use of a strain with such properties, as a biocatalyst, minimises the possibility of extraction of any toxic metabolites with the obtained product [44]. For these reasons, we decided to check the progress of biotransformation during the first day of substrate incubation, and the ability of this strain to hydrogenate higher concentrations of tested compounds. At this stage, the four most efficiently convertible compounds were used as substrates (1-4). The standard concentration of substrate for screening tests was 100 mg/L. However, we tested and compared the efficiency of this reaction by increasing the scale of the process so that the medium contained a maximum of 5 g of substrate per 1 L of a medium, which is shown in the table (Table 3). In this part of the experiment, the scale of the process was changed and Riplate square wells were used instead of Erlenmeyer flasks. This test was performed in triplicate. The substrate addition was as follows: 0.3, 1.5, 3, 6, and 15 mg per well, which corresponds to concentrations: 100, 500, 1, 2, and 5 g per 1 L of culture medium, respectively. At the lower concentrations, there is no significant difference between the conversion of various substrates. As the concentration increases, 2 -hydroxy-2"-methoxychalcone (1) is transformed the most efficiently, and the conversion is shown in Figure 2B. The lowest conversion rate recorded for this substrate after 24 h was 50%, at the maximum concentration of 5 g/L. As the concentration increased, significant differences between the conversion of individual compounds started to be visible ( Figure 3). The expected product (dihydrochalcone) was also observed during the transformation of the highest substrate concentration but with a much lower yield compared to the screening concentration (100 mg/L). After 24 h of biotransformation, at the highest substrate concentration (5 g/L), 2′-hydroxy-2″-methoxychalcone was converted the most efficiently-where about 50% conversion was observed ( Figure 3A). Under the above conditions, the Yarrowia lipolytica KCh 71 strain was also able to transform the other substrates, although with much lower yield (7%-22%) ( Table 3). In addition, such a high level of conversion for the tested methoxychalcones, at a concentration of 100 mg/L, is comparable to the previously described studies As the concentration increased, significant differences between the conversion of individual compounds started to be visible (Figure 3). The expected product (dihydrochalcone) was also observed during the transformation of the highest substrate concentration but with a much lower yield compared to the screening concentration (100 mg/L). After 24 h of biotransformation, at the highest substrate Catalysts 2020, 10, 1135 7 of 13 concentration (5 g/L), 2 -hydroxy-2"-methoxychalcone was converted the most efficiently-where about 50% conversion was observed ( Figure 3A). Under the above conditions, the Yarrowia lipolytica KCh 71 strain was also able to transform the other substrates, although with much lower yield (7%-22%) ( Table 3). In addition, such a high level of conversion for the tested methoxychalcones, at a concentration of 100 mg/L, is comparable to the previously described studies on the unsubstituted [13] or containing hydroxyl or methyl groups [47] chalcone derivatives.
lower yield (7%-22%) ( Table 3). In addition, such a high level of conversion for the tested methoxychalcones, at a concentration of 100 mg/L, is comparable to the previously described studies on the unsubstituted [13] or containing hydroxyl or methyl groups [47] chalcone derivatives.
Moreover, the use of yeast from the Yarrowia lipolytica species is not accidental. This is a microorganism whose properties are widely used in the food industry-from the production of citric acid [25]or eicosapentaenoic acid [48] by preventing rotting of the harvest [49] and cleaning the soil from petroleum hydrocarbons [50], to the production of high-protein animal feed [51] and unconventional food for humans [44]. It is also used in the production of various types of sweeteners [41]. Moreover, yeast of this species can be successfully used to produce significant amounts of various types of dihydrochalcones. In addition, biotransformations using these microorganisms allow one to obtain with high efficiency a single compound with high purity. The only reaction products were dihydrochalcones (also observed in our previous studies) [23]. The microorganisms used were not able to produce other flavonoid derivatives (flavones, flavanones), which are found during the transformation of chalcones in cultures of filamentous fungi, bacteria, or algae [13,47,52,53].  Moreover, the use of yeast from the Yarrowia lipolytica species is not accidental. This is a microorganism whose properties are widely used in the food industry-from the production of citric acid [25] or eicosapentaenoic acid [48] by preventing rotting of the harvest [49] and cleaning the soil from petroleum hydrocarbons [50], to the production of high-protein animal feed [51] and unconventional food for humans [44]. It is also used in the production of various types of sweeteners [41]. Moreover, yeast of this species can be successfully used to produce significant amounts of various types of dihydrochalcones. In addition, biotransformations using these microorganisms allow one to obtain with high efficiency a single compound with high purity. The only reaction products were dihydrochalcones (also observed in our previous studies) [23]. The microorganisms used were not able to produce other flavonoid derivatives (flavones, flavanones), which are found during the transformation of chalcones in cultures of filamentous fungi, bacteria, or algae [13,47,52,53].

Screening
Erlenmeyer flasks of 300 mL were used for biotransformation on an analytical scale, each containing 100 mL of Sabouraud culture medium (3% glucose, 1% aminobac). Used microorganisms were incubated for three days at 24 • C on a rotary shaker (144 rpm)(Eppendorf AG, Hamburg, Germany). After this time, 10 mg of the substrate was dissolved in DMSO (dimethyl sulfoxide) and added. Samples were collected after 1, 3, and 7 days. Portions of 10 mL of the transformation mixture were taken out and extracted with ethyl acetate. The extracts were dried over MgSO 4 , concentrated in vacuo, and analysed by gas chromatography (GC) and thin-layer chromatography (TLC) (SiO2, DC Alufolien Kieselgel 60 F254 (0.2 mm thick), Merck, Darmstadt, Germany).

Gas Chromatography
GC analysis was performed using an Agilent 7890A gas chromatograph, equipped with a flame ionisation detector (FID) (Agilent, Santa Clara, CA, USA). The capillary column DB-5HT (30 m × 0.25 mm × 0.10 µm) was used to determine the composition of the product mixtures. The temperature programme was applied as follows: 80-300 • C, the temperature on the detector: 300 • C, injection 1 µL, flow 1 mL/min, flow H2: 35 mL/min, airflow; 300 mL/min, time of analysis: 18.67 min. The retention times of the substrates and products are described in Table 4.

Preparative Scale
Preparative biotransformations were performed in 2 L Erlenmeyer flasks, each containing 500 mL of culture medium (3% glucose, 1% peptone). The transferred microorganisms were incubated for three days at 24 • C on a rotary shaker. After this time, 100 mg of the substrate dissolved in 2 mL of DMSO was added. After three days, the product was isolated by triple extraction with ethyl acetate (3 extractions with 300 mL), dried with anhydrous magnesium sulfate, and concentrated in vacuo. The transformation products were separated by preparative TLC and analysed (TLC, GC, NMR).

TLC and NMR Analysis
The course of biotransformation was monitored using TLC plates (SiO 2 , DC Alufolien Kiesel gel 60 F254 (0.2 mm thick), Merck, Darmstadt, Germany). Products were separated using preparative TLC plates (Silica Gel GF, 20 × 20 cm, 500 µm, Analtech, Newark, DE, USA) and a cyclohexane: ethyl acetate mixture (9:1, v/v) as an eluent, according to the method described previously [58]. The product was observed (without additional visualisation) under the UV lamp at the wavelength of 254 nm.

Increasing the Concentrations of Tested Substrates
Transfer (scaling) of the biotransformation scale was carried out in Riplate square wells, 24 wells, to which 3 mL of culture medium with an inoculum of Yarrowia lipolytica KCh 71 was added. Microorganisms were incubated for three days at 24 • C on an oscillating shaker (190 rpm)(ELMI, Riga, Latvia). Screening was performed for five substrate concentrations-100 mg/L, 500 mg/L, 1 g/L, 2 g/L, and 5 g/L. The substrate was dissolved in DMSO. A total of 100 µL of the prepared substrate solution was added to each well. The experiment was carried out in triplicate. Samples (500 µL each) were collected after 1, 3, 6, 12, and 24 h of substrate incubation, then extracted with ethyl acetate, centrifuged, and analysed with GC.

Conclusions
On account of the constantly growing requirement for sweeteners, new solutions for their production are still being sought, preferably as cheaply as possible, biotechnological and ones that simultaneously fulfil the assumptions of the "green source" theory. The use of microorganisms, which is approved as safe and even allowed for consumption, seems to be an interesting solution. Y. lipolytica KCh 71 adapted for biotransformation turned out to be the best of the tested microorganisms to transform methoxychalcones. The most efficiently transformed compounds were chalcones containing single methoxyl groups in the B ring. Interestingly, the substrate with 2 -hydroxy-2"-methoxychalcone even at a scale increased to 5 g substrate per 1 L of medium was convertible with up to 50% yield after 24 h. At the same time, a very high conversion, from 66 to 91% depending on the substrate, after one hour of incubation (Table 2) indicates that the ene-reductase catalysing this process is a constitutive enzyme. However, identification of which group of enzymes catalyzes this reaction requires the use of molecular biology methods.
With the increase in the number of methoxyl groups, the efficiency of the process decreased. For compounds that contain methoxyl substituents also in the A ring, the transformation did not occur at all, including commonly described flavokavain B (2 -hydroxy-4 ,6 -dimethoxychalcone).
The challenge facing this type of research is to limit the number of solvents used when extracting products-or to eliminate extraction. This solution would increase the efficiency of the process and, at the same time, would allow the production of a supplement containing the pro-health microorganism as well as a sweet flavonoid product with a significantly different spectrum of activity. Moreover, the use of created dihydrochalcones as sweeteners could have a positive effect on the human body, while enriching our diet and reducing the need to use sucrose and preventing various types of civilisation diseases.
Part of the 13 C NMR spectral of 1-(2 -hydroxyphenyl)-3-(2 ,5 -dimethoxyphenyl)-2-propan-1-one (12)  Author Contributions: M.Ł. performed the synthesis and biotransformations, interpreted the results, analyzed the spectral data, visualization, data curation, writing, original draft preparation; E.K. methodology, validation, reviewing and editing; E.K.-S. methodology, supervision, interpreted the results, analyzed the spectral data; T.J. conceptualization, validation, interpreted the results, analyzed the spectral data; Writing, reviewing and editing. All authors have read and agreed to the published version of the manuscript.
Funding: This study was financially supported by the Faculty of Biotechnology and Food Science, Wrocław University of Environmental and Life Sciences, grant number N070/0012/20.

Conflicts of Interest:
The authors declare no conflict of interest.

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