Heterologous production of equol by lactic acid bacteria strains in culture medium and food

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Introduction
Isoflavones are flavonoids present in various plants, particularly in soybean germ (Aguiar et al., 2007).They are classified as phytoestrogens since their structures resemble that of estrogen and have weak affinity for the estrogen receptor (Vaya and Tamir, 2004).Due to their biological activity, isoflavones have been linked to beneficial effects in health, including positive effects in ameliorating menopause symptoms and reducing the risk of cardiovascular disease and certain types of cancer (Ko, 2014;Mayo et al., 2019).Isoflavones are usually found in nature in their glycosylated form, which are not absorbed on the intestine, being daidzin and genistin the most abundant in soy.Glycosides must be hydrolyzed to aglycones (daidzein and genistein), by the appropriate glycosidases, to become bioavailable and physiologically active (Gaya et al., 2017;Setchell et al., 2002).Then, daidzein and genistein can be converted into dihydrodaidzein (DHD) and dihydrogenistein (DHG) by means of hydrogenation reaction.After this, the compounds O-desmethylangolensin (O-DMA) and/or equol can be formed from DHD by means of ring-cleavage or keto-elimination reactions, respectively.In the same way, DHG can be transformed into 6hydroxy-O-DMA or 5-hydroxy-equol (Gaya et al., 2018).
Equol is the isoflavone derivate with the highest estrogenic and antioxidant activity, it is characterized by being more stable than its isoflavone precursor (Lephart, 2019) and thus, there is an increasing interest in unraveling its influence in health (Mayo et al., 2019).Nevertheless, only a 30-50% of the western adult population produce equol, while production of O-DMA is much more extended (Atkinson et al., 2005).On its part, 5-hydroxy-equol, has been less studied, although it has more antioxidant activity than its precursor genistein (Arora et al., 1998) and has shown anticarcinogenic activity in vitro (Gao et al., 2018).
The interesting properties of equol have led to the searching of equolproducing bacteria of intestinal origin.In addition to bacterial combinations, strains capable of completing the transformation of daidzein into equol have been identified, mainly belonging to the family Eggerthellaceae, such as Adlercreutzia equolifaciens, Assacharobacter celatus, Eggerthella spp., Enterorhabdus mucosicola, Slackia isoflavoniconvertens and Slackia equolifaciens (Mayo et al., 2019).Besides these species, some intestinal lactic acid bacteria (LAB) strains have been described as equolproducers, i.e.Lactobacillus intestinalis (Heng et al., 2019) and Lactococcus garvieae 20-92 (Uchiyama et al., 2007).However, to date no bacteria with biotechnological interest, such as the LAB species commonly used as food fermentation starters or as probiotics, have been found able to produce equol from daidzein.
The enzymes involved in equol synthesis from daidzein are the daidzein reductase (DZNR), the DHD reductase (DHDR) and the tetrahydrodaidzein reductase (THDR) (Wang et al., 2005).A DHD racemase (DDRC) has been also described to play an important role in the production of equol, since the preferred substrate of DHDR is S-DHD but the main product of DZNR seems to be R-DHD and, thus, the racemase activity transforming the enantiomers favors the equol production (Shimada et al., 2012).The genes encoding those enzymes have been described to be organized in a cluster with similar sequence and genetic organization between different equol-producing strains such as S. isoflavoniconvertens DSM 22006, Eggerthella sp.YY7918 and L. garvieae 20-92 (Kawada et al., 2016).
The objective of this work was to produce equol and 5-hydroxy-equol by bacteria suitable for their use in fermented food, such as species of LAB generally recognized as safe (GRAS) and with qualified presumption of safety (QPS) status.The DZNR gene (dzr) had been previously cloned into different LAB and Bifidobacterium strains observing DHD and DHG production in fermented soy beverage (Peirotén et al., 2020b).In this work, the DHDR gene (ddr), THDR gene (tdr) and DDRC gene (ifcA) were cloned and transformed into different LAB strains, including the species Lactococcus lactis, Limosilactobacillus fermentum (previously Lactobacillus fermentum), Lacticaseibacillus casei (previously Lactobacillus casei) and Lactiplantibacillus plantarum (previously Lactobacillus plantarum).The production of equol and 5-hydroxy-equol from different precursors in culture medium and in a food matrix was studied.

Cloning of genes involved in the transformation of daidzein to equol from S. isoflavoniconvertens
Based on the published genome of S. isoflavoniconvertens DSM 22006, specific primers (Table 2) were designed to amplify the genes dzr, ddr, tdr and ifcA.The genes ddr and tdr were amplified together in a single amplicon using the F-tdr and R-ddr oligonucleotides (Fig. 1).The forward F-tdr primer introduced a BspHI restriction site around the initiation codon of the THDR gene, and the reverse R-ddr primer introduced an XbaI site downstream of the stop codon of the DHDR gene.The PCR product was digested with the same restriction enzymes and ligated into the vector pNZ:TuR (Landete et al., 2015), previously digested with XbaI and NcoI, which produces BspHI compatible cohesive ends, to obtain the plasmid, pNZ:TuR.tdr.ddr.
The gene encoding DDRC (ifcA) was amplified by PCR using F-rac and R-rac primers (Table 2).Additionally, ifcA, was amplified together with the adjacent genes ifcB and ifcC, which are located downstream from ifcA and upstream of tdr in the S. isoflavoniconvertens genome (Fig. 1), by using the F-rac and R-ifcABC oligonucleotides.The forward primer F-rac introduced a PciI site before the initiation codon of ifcA, and the reverse primers R-rac and R-ifcABC introduced an XbaI site downstream of the stop codon of ifcA and ifcC respectively.The PCR products were digested with the two restriction enzymes and ligated into the vector pNZ:TuR (Landete et al., 2015), previously digested with XbaI and NcoI, which produces PciI compatible cohesive ends.The resulting plasmids were pNZ:TuR.ifcAand pNZ:TuR.ifcABC.Finally, joint cloning of dzr, ddr and tdr was attempted by using the primers F-tdr and R-DaidR.
The constructed plasmids were individually transformed into L. lactis MG1363 by electroporation (Landete et al., 2014); transformants were selected in GM17 agar with chloramphenicol (5 μg/mL, Merck KGaA) and checked by restriction mapping and sequencing the inserted fragment.L. lactis MG1363 was the host organism for subsequent transformations of L. casei BL23, L. plantarum WCFS1, L. fermentum INIA 584L  Gasson (1983) and L. fermentum INIA 832L with each of the plasmids (Table 1).These other LAB strains were transformed by electroporation followed by selection in MRS agar with chloramphenicol (5 μg/mL).
Moreover, a series of co-cultures were performed in the same conditions described above, pairing couples of strains harbouring the genes of the reductases DHDR and THDR (pNZ:TuR.tdr.ddr) on one side, and the gene encoding DDRC (either the pNZ:TuR.ifcAor pNZ:TuR.ifcABC) on the other.

in culture medium and cow's milk
Three different combinations of the transformants of each LAB were co-cultivated (1% v/v, between 1 × 10 7 and 1 × 10 8 cfu/mL) in BHI medium, supplemented with daidzein (50 mg/L; 196.77 μM) and genistein (50 mg/L; 185.02 μM) (LC Laboratories, Woburn, MA, USA) and incubated at their optimal temperature (30 • C or 37 • C), for 72 h under anaerobic conditions.Parental strains cultivated in the same conditions were used as controls.For each strain, different combinations were made of the strains harbouring i) pNZ:TuR.dzr(Peirotén et al., 2020b) and pNZ:TuR.tdr.ddr;ii) pNZ:TuR.dzr,pNZ:TuR.tdr.ddr and pNZ:TuR.ifcA;iii) pNZ:TuR.dzr,pNZ:TuR.tdr.ddr and pNZ:TuR.ifcABC.S. isoflavoniconvertens DSM22006 was grown in Wilkins-Chalgren broth supplemented with the same amounts of daidzein and genistein, and was incubated at 37 • C under anaerobic conditions.Moreover, L. fermentum INIA 584L and L. fermentum INIA P832L were subjected to a similar incubation using cow's milk (reconstituted cow skim milk prepared according to the manufacturer's specifications, BD Biosciences) as matrix for the metabolization of daidzein and genistein, which were added in the same concentrations as before.The combinations used in this experiment for each of the strains were i) pNZ:TuR.dzrand pNZ:TuR.tdr.ddr;ii) pNZ:TuR.dzr,pNZ:TuR.tdr.ddr and pNZ:TuR.ifcA.Parental strains were again used as controls.Incubations were carried out for 72 h under anaerobic conditions at 37 • C.

Extraction of isoflavones from culture medium
After incubations of the parental and transformed LAB in the combinations and conditions described above, isoflavones were extracted from the culture medium twice with 2 mL of diethyl ether and twice with 2 mL of ethyl acetate, according to Gaya et al. (2016a).The solvents were evaporated at room temperature under a N 2 stream, and the residue was dissolved in 300 μL methanol/water (50:50, v/v).Filtering, through a 0.22 μm cellulose acetate filter (Millipore, Madrid, Spain), was performed before transferring the extracts to HPLC vials and storing them at − 20 • C.

Extraction of isoflavones from cow's milk
After incubation in cow's milk of the different combinations of LAB  described in Section 2.4., isoflavones were extracted following the official AOAC method (Collison et al., 2008).Briefly, 1 mL of the sample was mixed with 500 μL of acetonitrile and 150 μL of H 2 O, shaken vigorously for 60 min and centrifuged for 10 min at 13000 rpm.The supernatant was filtered through a 0.22 μm PFTE membrane (Whatman; Cytiva, Little Chalfont, UK), and stored at − 20 • C.

Identification and quantification of isoflavones
Isoflavones were analyzed by HPLC-ESI/MS as described in Gaya et al. (2016a) and Gaya et al. (2016b).Quantification was carried out by means of external standard calibration curves of equol and the precursors DHD, DHG, daidzein and genistein (LC Laboratories, Woburn, MA, USA).Stock solutions (10 mg/L) of polyphenols were prepared in DMSO (Merck KGaA).5-hydroxy-equol was not available as a standard in any commercial house.The incubation of genistein and DHG with S. isoflavoniconvertens DSM22006 allowed us to identify the 5-hydroxyequol peak by HPLC-ESI/MS analysis.So, 5-hydroxy-equol was quantified using the calibration curve of the more similar compound, equol.

Statistical analysis
Statistical analysis of the isoflavones concentration was performed using the SPSS Statistics 22.0 software (IBM Corp., Armonk, NY, USA).Data were analyzed by ANOVA using a general lineal model (GLM).Comparison of means was carried out by Tukey test, with a confidence interval of 99%.
All the LAB strains harbouring pNZ:TuR.tdr.ddr,encoding DHDR and THDR, were able to produce equol from DHD (Table 3).Nevertheless, the yields in equol production varied greatly between strains.The two L. fermentum strains consumed DHD in similar levels to S. isoflavoniconvertens and showed the highest production among the recombinant LAB strains, transforming nearly half of the DHD added to the medium into equol, although they did not reach the levels of production of S. isoflavoniconvertens.The rest of LAB harbouring pNZ:TuR.tdr.ddr showed lower metabolization of DHD and smaller amounts of equol produced.On its part, 5-hydroxy-equol was only produced by the L. fermentum strains and with lower yields than the ones obtained with equol.Interestingly, although the two L. fermentum strains harbouring pNZ:TuR.tdr.ddrconsumed more DHG than S. isoflavoniconvertens, this had no reflection in more 5-hydroxy-equol production.
The co-incubation of the LAB strains harbouring pNZ:TuR.tdr.ddr with the correspondent strains containing DDRC, harbouring either pNZ:TuR.ifcAor pNZ:TuR.ifcABC,did not result in significant changes in the equol and 5-hydroxy-equol production from DHD and DHG in BHI medium (Table 3).

Equol and 5-hydroxy-equol production from daidzein and genistein in culture medium by recombinant LAB strains
After corroborating the transformation of DHD and DHG by LAB strains harbouring pNZ:TuR.tdr.ddr,new co-incubations with daidzein and genistein as precursors were carried out.In this case, the co-cultures included the strains harbouring pNZ:TuR.dzr,which in previous works had shown to reduce daidzein and genistein into DHD and DHG (Peirotén et al., 2020b).All the strains combinations, i.e. harbouring the dzr gene and the combined ddr and tdr genes, resulted in an efficient transformation of daidzein into DHD and of genistein into DHG, and in the production of equol (Table 4).Once more, the combinations of the recombinant L. fermentum strains showed the highest production of equol and were the only ones producing 5-hydroxy-equol.Conversely, the correspondent parental strains showed only small amounts of DHD and no production of equol, DHG or 5-hydroxy-equol.
Once the production of equol and 5-hydroxy-equol was demonstrated by the heterologous expression of the genes dzr, ddr and tdr in L. fermentum strains, we worked on the joint cloning of ddr, tdr and dzr with primers F-tdr and R-Daidr (Fig. 1).However, although we achieved amplification, we did not obtain transformants and thus, the transformation of daidzein and genistein by sole strains expressing simultaneously the three genes could not be tested.
The addition of the DDRC to the co-cultures, i.e. incubation of triplets of strains harbouring pNZ:TuR.dzr,pNZ:TuR.tdr.ddr and pNZ:TuR.ifcATable 3 Production of equol and 5-hydroxy-equol from DHD (195.12 μM) and DHG (183.65  or pNZ:TuR.ifcABC,caused and increment on the equol production in the two L. fermentum strains, which showed a high level of DHD transformation into this metabolite, reaching an 80-87% of transformation of the daidzein into equol and surpassing the yields of equol production by S. isoflavoniconvertens DSM 22006 (Table 4).A similar tendency was observed regarding the production of 5-hydroxy-equol, which showed a slight increase in the two L. fermentum strains when ifcA was present.In the rest of recombinant LAB there was no significant changes, except for a slight increase of DHD in the case of L. lactis MG1363 when strains harbouring pNZ:TuR.ifcAor pNZ:TuR.ifcABCwere added to the medium.

Equol and 5-hydroxy-equol production by L. fermentum strains in cow's milk supplemented with daidzein and genistein
In order to seek the applicability of the recombinant LAB strains in the development of functional foods enriched in equol, the two L. fermentum strains were incubated in cow's milk supplemented with daidzein and genistein.
The different combinations of recombinant strains showed similar behavior to that observed in culture medium, showing high metabolization of daidzein and genistein and the production of equol and 5-hydroxy-equol in the supplemented milk (Table 5).For those strains, the addition of the strain harbouring pNZ:TuR.ifcA to the mixture of recombinant strains resulted in a clear increase of the equol yields, with around four times more transformation of the initial daidzein into equol.

Discussion
It has been widely demonstrated that LAB strains are able to deglycosylate glycoside isoflavones in culture media and beverages (Gaya et al., 2016c;Rekha and Vijayalakshmi, 2011).Even, some LAB strains are capable of producing high concentrations of daidzein and genistein in soy beverages (Delgado et al., 2019;Peirotén et al., 2020a).However, equol production has been mainly described within the family Eggerthellaceae (Mayo et al., 2019), while LAB strains completing the transformation of daidzein and genistein are scarce, and those described (Heng et al., 2019;Uchiyama et al., 2007) belong to species without a tradition of usage in food fermentations and not included in the GRAS and QPS lists.The heterologous production of equol and 5-hydroxyequol has been previously explored by means of recombinant E. coli strains expressing the enzymes involved in the metabolic transformation of daidzein (Kawada et al., 2016;Lee et al., 2016).In this work, we have explored the viability of heterologous production of equol by different LAB belonging to species commonly used in food fermentation and thus GRAS.
The joint cloning of ddr and tdr from S. isoflavoniconvertens allowed the transformation of DHD into equol by recombinant LAB belonging to the L. lactis, L. casei, L. plantarum and L. fermentum species (Table 3), while the addition of dzr resulted in the production of equol from daidzein (Table 4) in agreement with that described in recombinant E. coli (Tsuji et al., 2012).Interestingly, the two recombinant L. fermentum strains showed production, both from DHG and genistein, of 5-hydroxy-equol, whose production by recombinant E. coli has been scarcely reported (Lee et al., 2017).In addition, it was observed how the combination of DHDR and THDR led to a less efficient transformation of DHG than DHD and, therefore, the 5-hydroxy-equol production was much lower than the equol production.These results are in accordance with the lack of production of 5-hydroxy-equol by recombinant E. coli (Schröder et al., 2013) and the less efficient formation of 5-hydroxyequol in comparison to equol observed in vitro and in vivo with intact cells of S. isoflavoniconvertens (Matthies et al., 2009).
The addition of the strains harbouring ifcA resulted in an increment of equol production from daidzein only in the case of the two L. fermentum strains.Similarly to the results of Shimada et al. (2012), the addition of the recombinant DDRC resulted in a 3.6 to 3.9 fold increase of equol production by the L. fermentum strains.The lack of activity of DDRC in the rest of the LAB strains tested, reflected in no increment of equol production, could be related to a lower activity of the enzymes DHDR and/or THDR in those strains, since they also produce less equol than the L. fermentum strains (Table 4), although other factors may be affecting.The apparent lack of activity of DDRC in L. fermentum when incubated with DHD is likely due to this commercial compound being a racemic mixture, while the DHD produced by DZNR from daidzein has been described to be mainly R-DHD (Shimada et al., 2012).Given also the described affinity of DHDR for S-DHD, the action of this racemase would be of importance when DHD is produced mainly as the R enantiomer, but not when using a racemic mixture as substrate.The importance of DDRC for an efficient production of 5-hydroxyequol has been also described (Lee et al., 2017).In this work, we found a slight increase on 5-hydroxy-equol production from genistein by the two L. fermentum strains upon the addition of the strains with the recombinant DDRC, although not statistically significative.Conversely, S. isoflavoniconvertens showed fewer production of equol from daidzein compared to DHD as precursor, achieving lower equol concentrations than the combination of L. fermentum strains harbouring ifcA (Table 4).In this regard, since both DZNR and DHDR need NADPH as cofactor, the transformation of daidzein could be interfering with the transformation of DHD, as compared with the transformation for DHD directly in which the NADPH is used only by DHDR.This could have a higher impact on S. isoflavoniconvertens since the reactions take place within the same cell, while the reactions are compartmentalized in L. fermentum strains, i.e. daidzein is reduced by L. fermentum pNZ:TuR.dzrwhile DHD is transformed by L. fermentum pNZ:TuR.tdr.ddr.This beneficial effect of compartmentalization in equol production has been described previously in recombinant E. coli (Lee et al., 2017).
The genes ifcB and ifcC, which are located between the genes ifcA and tdr in the S. isoflavoniconvertens cluster, were tested together with ifcA, resulting in no differences between adding the strains with just ifcA (pNZ:TuR.ifcA)or with the three genes (pNZ:TuR.ifcABC).This suggests that those two genes seem to have no influence in the equol production under the conditions tested.
S. isoflavoniconvertens produced higher concentration of equol and 5hydroxy-equol than the majority or the different co-cultures of transformed LAB strains incubated with isoflavone precursors in medium (Tables 3 and 4).However, L. fermentum INIA 584L and L. fermentum INIA 832L harbouring the combination of dzr, ifcA, tdr and ddr surpassed the production of equol from daidzein showed by S. isoflavoniconvertens DSM22006, and equaled its production of 5-hydroxy-equol from genistein (Table 4).
Taking into account the equol production by the L. fermentum strains, we assayed the production of equol in a food matrix.We chose a food matrix with no isoflavones in its composition (cow's milk), and supplemented it with known amounts of daidzein and genistein.Similarly to the results with culture medium, the combination of the four genes, dzr, ifcA, tdr and ddr, resulted in a highly efficient production of equol in the incubated milk (Table 5).The use of soy beverage as base for this kind of product should take into account that it has a complex composition of isoflavones and that they are present mainly in the form of glycosides.Thus, in order to obtain an efficient production of equol, the glycosidase activity should be ensured in the form of another bacterial strain with high glycosidase activity or of a recombinant strain harbouring an efficient β-glucosidase (Gaya et al., 2020).
LAB strains expressing genes of interest have great potential in the development of functional products, such as food enriched in equol.Nevertheless, safety and legal issues must be taken into account in order to obtain an authorization, which, in the EU follows a case-by-case approach (EFSA, 2011) that has put the use of this kind of microorganisms under a de facto moratorium.Within this scenario, the products with transformed bacteria undergo less restrictive requirements if the bacteria and its DNA is not present in the final product.Seeing this, the more feasible alternative for a soy beverage enriched in equol and 5-hydroxy-equol by the fermentation of recombinant LAB strains would have to incorporate a later treatment to eliminate the bacteria and their DNA.

Conclusions
This is the first report concerning the use of recombinant bacteria with interest in food, such as LAB belonging to GRAS species, to produce equol and 5-hydroxy-equol.The combination of L. fermentum strains harbouring the S. isoflavoniconvertens enzymes DZNR, DDRC, DHDR and THDR showed efficient transformation of daidzein to equol, both in culture medium and in milk.This opens the possibility for the development of fermented foods enriched in equol.

Table 1
Bacterial strains and plasmids used in this work.

Table 2
Plasmids used in this work.
μM) by parental and transformed strains of LAB and S. isoflavoniconvertens DSM22006 in culture medium.
A. Ruiz de la Bastida et al.

Table 4
Metabolism of daidzein (196.77μM)andgenistein (185.02μM) by parental and transformed LAB strains and S. isoflavoniconvertens DSM22006 in culture medium.Values in the same column with different superscript differ significantly (P < 0.01); n.d.not detected.
a-d Values in the same column with different superscript differ significantly (P < 0.01); n.d.not detected.