Amelia PDF

AEM Accepted Manuscript Posted Online 18 May 2018
Appl. Environ. Microbiol. doi:10.1128/AEM.01123-18

Copyright © 2018 American Society for Microbiology. All Rights Reserved.
2 Unravelling the reduction pathway as alternative metabolic
3 route to hydroxycinnamate decarboxylation in Lactobacillus
Downloaded from http://aem.asm.org/ on September 22, 2019 by guest

4 plantarum
8 Laura Santamaría, Inés Reverón, Félix López de Felipe, Blanca de las Rivas, Rosario
9 Muñoz
10
11
12 Laboratorio de Biotecnología Bacteriana, Instituto de Ciencia y Tecnología de Alimentos y
13 Nutrición, ICTAN-CSIC, Madrid, Spain
14
15
16
17 Address correspondence to Rosario Muñoz, rmunoz@ictan.csic.es
18
19
20
21 Running Title
22 LACTOBACILLUS PLANTARUM HYDROXYCINNAMATE REDUCTASE
1
23 ABSTRACT
24 Lactobacillus plantarum is the lactic acid bacterial species most frequently found in
25 plant-food fermentations where hydroxycinnamic acids are abundant. L. plantarum
26 efficiently decarboxylates these compounds, and also reduces them, yielding

27 substituted phenylpropionic acids. Although the reduction step is known to be
28 induced by a hydroxycinnamic acid, the enzymatic machinery responsible for this
29 reduction pathway has not been yet identified and characterized. A previous study on
30 the transcriptomic response of L. plantarum to p-coumaric acid revealed a marked
31 induction of two contiguous genes lp_1424 and lp_1425, encoding putative reductases.
32 In this work, disruption of these genes abolished the hydroxycinnamate reductase
33 activity of L. plantarum, supporting their involvement in such chemical activity.
34 Functional in vitro studies reveal that Lp_1425 (HcrB) exhibits hydroxycinnamate
35 reductase activity but was unstable in solution. In contrast, Lp_1424 (HcrA) was
36 inactive but showed high stability. When the hcrAB genes were co-overexpressed,
37 formation of an active heterodimer (HcrAB) was observed. Since L. plantarum
38 reductase activity was only observed on hydroxycinnamic acids (o-coumaric, m-
39 coumaric, p-coumaric, caffeic, ferulic, and sinapic acids), the presence of a hydroxyl
40 group substituent on the benzene ring appears to be required for activity. In addition,
41 hydroxycinnamate reductase activity was not widely present among lactic acid
42 bacteria, and it was associated to the presence of hcrAB genes. This study revealed
43 that L. plantarum hydroxycinnamate reductase is a heterodimeric NADH-dependent
44 coumarate reductase acting on a carbon-carbon double bond.
45
2
46 IMPORTANCE
47
48 Lactobacillus plantarum is a bacterial species frequently found in the fermentation of
49 vegetables where hydroxycinnamic acids are present. The bacterial metabolism on

50 these compounds during fermentation plays a fundamental role on the biological
51 activity of hydroxycinnamates. L. plantarum strains exhibit an as yet unknown
52 reducing activity, transforming hydroxycinnamates to substituted phenylpropionic
53 acids, which possess higher antioxidant activity that their precursors. The protein
54 machinery involved in hydroxycinnamate reduction, HcrAB, was genetically
55 identified and characterized. The heterodimeric NADH-dependent coumarate
56 reductase HcrAB described in this work provides new insights on the L. plantarum
57 metabolic response to counteract the stressful conditions generated by food phenolics.
58
3
59 INTRODUCTION
60 Phenolic compounds in foods have attracted great interest due to growing evidence of their
61 beneficial effect on human health. Phenolic acids account for almost one third of the dietary
62 phenols and are associated with organoleptic, nutritional, and antioxidant properties of

63 foods. Hydroxycinnamic acids, such as p-coumaric, caffeic, ferulic and sinapic acids, are
64 the most abundant class of phenolic acids in fruits, vegetables, and cereal grains (1). These
65 hydroxycinnamic acids could be transformed during the fermentation of plant-based foods.
66 Fermentative microbiota plays an important role in the biological activity of these acids and
67 therefore the elucidation of their bacterial metabolism will provide important clues on their
68 beneficial effects.
69 Lactic acid bacteria are widely present in food fermentations, and L. plantarum is
70 the species most frequent found in plant fermentations where phenolic compounds are
71 abundant (2). Previous studies on L. plantarum demonstrated the existence of two
72 metabolic pathways for the transformation of hydroxycinnamates. The most efficient
73 pathway entails the successive action of a phenolic acid decarboxylase (PDC) and a
74 reductase (3). In this pathway, p-coumaric, caffeic, and ferulic acid are decarboxylated by
75 the PDC action to their corresponding vinyl derivatives (vinylphenol, vinylcatechol, and
76 vinylguaiacol, respectively) (4, 5), which are subsequently reduced to the resultant ethyl
77 derivatives (ethylphenol, ethylcatechol, and ethylguaiacol).
78 The second metabolic pathway for the transformation of hydroxycinnamates in L.
79 plantarum implies the reduction of these acids (p-coumaric, m-coumaric, caffeic, and
80 ferulic acids) to their corresponding phenylpropionic substituted acids [phloretic, 3-(3-
81 hydroxyphenyl) propionic acid (3-HPPA), dihydrocaffeic, and dihydroferulic acids] (6).
82 Nonetheless, the reductase activity appeared to be about 100-fold lower than the
4
83 decarboxylase activity (3). The reduction pathway was more evident when a L. plantarum
84 mutant strain deficient on PDC activity was constructed (3). Thus, when PDC mutant
85 (Δlp_3665) cells were induced with p-coumaric acid, only phloretic acid was detected (3)
86 instead of 4-ethylphenol, which would result from a decarboxylation reaction. These results

87 demonstrated that the enzyme(s) involved in the reduction of hydroxycinnamates (such as
88 p-coumaric acid) were synthesized after their induction by the hydroxycinnamic acid (6).
89 Within this context, a previous study on the global transcriptomic response of L. plantarum
90 exposed to 1.5 mM p-coumaric acid stress (7) revealed that the most upregulated gene was
91 by far pdc (lp_3665), which was induced 112-fold as compared to the growth in absence of
92 this hydroxycinnamate. Interestingly, a marked induction of some other genes coding for
93 putative reductases was also detected. The oxidoreductase responsible of this second
94 pathway remains biochemically and genetically uncharacterized.
95 Presently, many oxidoreductases of the class EC 1.3.1 acting on carbon-carbon
96 double bonds in α, β-position to a carbonyl group have been described; however, only few
97 of them have been reported to work on hydroxycinnamic acids (8). The 2-coumarate
98 reductase subgroup (EC 1.3.1.11) only includes a NADH-dependent reductase partially
99 purified from Arthrobacter extracts which reduces o-coumaric acid to melilotic acid (9).
100 This enzyme was highly specific for o-coumarate, as the activities against cinnamic acid
101 and several substituted cinnamates (including p-coumaric, caffeic, ferulic and sinapic acids)
102 were very low (10). The subgroup of 2-enoate reductases (EC 1.3.1.31) also included
103 enzymes for the hydrogenation of C=C bond on hydroxycinnamates. Enoate reductases
104 belong to the rare class of flavoenzymes in which the involvement of iron-sulfur centers in
105 electron transfers has been well established (11). Enoate reductases are characterized by
106 their high stereospecificity, strict regioselectivity, and a rather broad substrate specificity
5
107 (12). These enzymes, which have been found in numerous Clostridia and other strict
108 anaerobes, have been scarcely studied most probably due to their high sensitiveness to
109 oxygen (13). The substrate specificity of clostridial enoate reductases is very variable,
110 reducing either a broad range of enoates (C.kluyveri and C. tyrobutyricum) or almost

111 exclusively cinnamic acid (C. sporogenes) (14). In this latter case, whereas p-coumaric acid
112 resulted a relatively good substrate for the enoate reductase from C. sporogenes, the
113 enzyme was almost inactive on caffeic acid (15). Other hydroxycinnamic acids were also
114 substrates for clostridial enoate reductases: p-coumaric acid was reduced by C.
115 acetobutylicum enoate reductase (8) and o-coumaric acid by Clostridium spec. La 1 protein
116 (13). Despite clostridia are phylogenetically related to lactobacilli, nowadays, the
117 oxidorreductase type acting on the hydroxycinnamate carbon-carbon double bond in L.
118 plantarum remain largely unknown.
119 In this work, the genes involved in L. plantarum hydroxycinnamate reduction have
120 been identified. In addition, in this study a hydroxycinnamate reductase belonging to the 2-
121 coumarate reductase subgroup (EC 1.3.1.11) has been genetically identified and
122 characterized.
123
124 RESULTS AND DISCUSSION
125
126 Identification of the enzyme responsible for hydroxycinnamate reduction in L.
127 plantarum WCFS1. Previous studies on the global transcriptomic response of L. plantarum
128 exposed to p-coumaric acid revealed a marked induction of genes coding for putative
129 reductases (7). Thus, the third most overexpressed transcript (19.7-fold induction) was
130 lp_1425, which would encode a fumarate reductase/succinate dehydrogenase, FAD-binding
6
131 flavoprotein NADPH-dependent FMN reductase. In addition, two other genes clustered
132 along with lp_1425, namely, lp_1424 (putative NADPH-dependent FMN reductase family
133 protein) and lp_1426 (protein of unknown function) were also strongly upregulated (15.3
134 and 7.2-fold, respectively). In order to determine if one (or both) of these genes encoding

135 the reductase(s) were involved in hydroxycinnamate reduction, lp_1424 and lp_1425
136 knockout mutants were constructed by an insertion-duplication strategy. The correct
137 insertion of the donor plasmids into the L. plantarum WCFS1 chromosome was verified by
138 PCR (see Materials and Methods section). The wild-type and mutant strains were grown in
139 MRS medium supplemented with 1.5 mM m-coumaric acid. After 10 days of incubation,
140 samples were collected, and the phenolic compounds present in the supernatants were
141 extracted and analysed by HPLC (Fig. 1). The results indicated that these two mutants were
142 unable to reduce m-coumaric acid, most probably because both proteins are required by L.
143 plantarum to reduce hydroxycinnamates. In this assay, m-coumaric acid was chosen as
144 model compound of hydroxycinnamic acid since L. plantarum is unable to decarboxylate it,
145 as it was reported previously (5), although it reduces this compound, yielding 3-HPPA (6).
146 These results suggested that the two consecutive genes lp_1424 (hcrA, hydroxycinnamate
147 reductase) and lp_1425 (hcrB) are needed for reducing m-coumaric acid.
148 Genetic organization and expression profile of the hcr locus on L. plantarum
149 WCFS1. In order to characterize in deeper detail the roles of hcrA and hcrB in the context
150 of hydroxycinnamate reduction, their genetic organization and expression profile in L.
151 plantarum WCFS1 were analysed (Fig. 2A). To determine the transcriptional profile of the
152 lp_1420-lp_1427 region, total RNA from L. plantarum WCFS1 cells grown in MRS
153 medium supplemented with p-coumaric acid (1.5 mM) was isolated and analysed by RT-
154 PCR (Table 1, Fig. 2B). To investigate the potential involvement of the lp_1422 and hcrC
7
155 genes in the reduction of hydroxycinnamates, their corresponding L. plantarum WCFS1
156 knockout mutants were constructed. Whereas the detection of reductase activity in the hcrC
157 mutant grown in the presence of m-coumaric acid indicated that HcrC plays no role (or an
158 ancillary one) in the reduction of hydroxycinnamates, the lack of activity in the knockout

159 lp_1422 mutant (Figure S1 in the supplemental material) supports the participation of
160 HcrR, a LysR-type transcriptional regulator, in the reduction process.
161 Moreover, since the transcriptional analysis showed co-transcription of the hcrA,
162 hcrB and hcrC genes (Fig. 2), it could be possible that disruption of hcrA may prevent the
163 induction of the other downstream, cotranscribed genes. This, in turn, could be the basis of
164 reductase activity abolition. Quantitative RT-PCR experiments demonstrates the presence
165 of polar effects on the hcrB transcription due to the disruption of the upstream hcrA gene
166 (Fig. 3).
167 Therefore, as a whole, it could be concluded that the presence of a functional HcrB
168 protein is a minimum requirement for hydroxycinnamate reductase activity in L. plantarum
169 since the hcrB knockout mutant, despite producing HcrA protein, lacks reductase activity.
170 Hydroxycinnamate reductase activity of HcrA and HcrB proteins. Once the
171 necessity of the HcrB protein has been determined for the reducing activity of L.
172 plantarum, it was determined whether this protein is sufficient for this bacterium to exhibit
173 such enzymatic activity in vitro. With this aim, the hcrA and hcrB genes were cloned either
174 individually or jointly, as they appeared on the L. plantarum WCFS1 chromosome. Details
175 about the cloning strategy can be found in Table 1. The correct sequences of the
176 recombinant plasmids pURI3-Cter-HcrA, pURI3-Cter-HcrB, and pURI3-Cter-HcrAB were
177 verified by DNA sequencing.
8
178 Cell extracts were prepared from E. coli BL21 cells harbouring the recombinant
179 plasmids. The pURI3-Cter-HcrA cell extract showed one major protein band of
180 approximately 24 kDa in SDS-PAGE, which was absent in control cells containing the
181 pURI3-Cter vector plasmid alone (Fig. S2A in the supplemental material). The molecular

182 mass of this overproduced protein is in good agreement with the one deduced from its
183 amino acid sequence as derived from the hcrA gene. These extracts were incubated in the
184 presence of 1.5 mM m-coumaric acid and the reaction products were extracted by ethyl
185 acetate and analysed by HPLC (Fig S2B). Similar to the extracts from control cells, those
186 with the HcrA protein contained m-coumaric acid but not the expected reduced product 3-
187 HPPA.
188 The HcrA protein was purified by immobilized metal affinity chromatography. The
189 purified protein showed the characteristic yellow colour indicative of the binding of an
190 oxidized flavin cofactor. However, similarly to cell extracts, when this pure HcrA protein
191 was added to a reaction mixture containing FMN (7.5 mM), and NADH or NADPH (15
192 mM) no reduction of m-coumaric acid was detected (Fig S2B).
193 On the other hand, SDS-PAGE analysis of pURI3-Cter-HcrB cell extracts showed
194 an intense 90 kDa protein band which was absent from control cells (Fig. 4A). The
195 molecular mass of this protein agrees well with the expected for HcrB (88 kDa). When
196 these extracts were incubated with 1.5 mM m-coumaric acid, it was observed that this
197 hydroxycinnamic acid was fully reduced to 3-HPPA. As was the case for HcrA, the purified
198 HcrB protein also showed a yellow colour indicative of a complex between the protein and
199 an oxidized form of flavin cofactors. When this protein was incubated with FMN (7.5 mM)
200 and NADH (15 mM), it was able to reduce the m-coumaric acid present in the in vitro
201 reaction (Fig. 4B).
9
202 Regarding the dependence of the reducing activity of m-coumaric acid by HcrB on
203 the cofactors used, control experiments revealed, as otherwise expected, lack of activity in
204 the absence of FMN or NADH (data not shown). On the other hand, no activity was
205 observed upon replacement of NADH with NADPH, indicating that the HcrB activity is

206 NADH-dependent. These results indicate that HcrB utilizes FMN and NADH as cofactors
207 to reduce m-coumaric acid to 3-HPPA, and that HcrA lacks this in vitro enzymatic activity.
208 m-Coumaric acid was reduced to 3-HPPA when pure HcrB protein was assayed,
209 although a major peak of m-coumaric acid remained (Fig 4B). However, protein extracts
210 from E. coli producing recombinant HcrB were able to reduce all the m-coumaric acid
211 present on the reaction mixture (Fig. 4B). Therefore, these cell extracts were used to assay
212 reductase activity against five additional hydroxycinnamic acids (p-coumaric, o-coumaric,
213 ferulic, caffeic and sinapic acids) and cinnamic acid (Fig. 5). A schematic representation of
214 these acids is shown in Fig. S3. All the hydroxycinnamic acids assayed were reduced to
215 their corresponding phenylpropionic acids, such as p-coumaric acid to phloretic acid, m-
216 coumaric acid to 3-HPPA, o-coumaric acid to melilotic acid or 3-(2-hydroxyphenyl)
217 propionic (2-HPPA), ferulic acid to hydroferulic acid, caffeic acid to hydrocaffeic, and
218 sinapic acid to hydrosinapic acid. The identification of these compounds was carried out by
219 comparing the retention times and spectral data of each peak with those of standards from
220 commercial suppliers. The study revealed that HcrB needs a hydroxyl group substituent on
221 the benzene ring for activity as reduction was only observed on hydroxycinnamates (o-
222 coumaric, m-coumaric, p-coumaric, caffeic, ferulic, and sinapic acids).
223 The acids reduced by HcrB are in agreement with previous results when the
224 metabolism of hydroxycinnamic acids was studied in L. plantarum cell cultures (6), except
225 for the o-coumaric acid metabolism, which was not observed. The reason for the absence of
10
226 o-coumarate reductase activity in L. plantarum cultures is currently unknown. Searching a
227 possible explanation for this difference, we studied the relative expression of all the
228 described hcr genes (hcrR, hcrA, hcrB, and hcrC) upon exposure to a hydroxycinnamic
229 acid (Fig. 6). The responses to the presence of cinnamic acid and gallic acid (an

230 hydroxybenzoic acid) were also included in the study. Unexpectedly, the expression results
231 did not explain the difference observed as hcrABC genes were induced in the presence of o-
232 coumaric as well as in response to other hydroxycinnamic acids. In spite that cinnamic acid
233 is not a substrate for the reductase, the hcrABC genes were also induced, possibly due to its
234 structural similarity. However, the studied genes were not induced in response to the
235 presence of a hydroxybenzoic acid (gallic acid).
236 Role of HcrA in reductase activity. The results obtained above support that HcrB
237 reduces hydroxycinnamates, and that HcrA lacks this enzymatic activity. Purified HcrB
238 protein in the presence of appropriate cofactors (FMN and NADH) reduces in vitro, only
239 partially, m-coumaric acid. In order to elucidate a possible role of HrcA in
240 hydroxycinnamate reductase activity, the hcrA and hcrB genes were also jointly cloned, as
241 they appeared on the L. plantarum WCFS1 chromosome. When cell extracts were prepared
242 from E. coli BL21 harbouring pURI3-Cter-HcrAB recombinant plasmid, it could be
243 observed the presence of the two expected bands (22 and 87 kDa) on SDS-PAGE, which
244 were absent on control cells containing the pURI3-Cter vector plasmid alone (Fig. 7A).
245 Since according to the cloning strategy of hcrAB in the pURI3-Cter plasmid, only the HcrB
246 protein has a His6 tag (at its C-terminal end), it was surprising to observe both proteins co-
247 eluting from the IMAC step (Fig. 7B) since it reveals that both proteins form a stable
248 complex.
11
249 Alignment of the amino acid sequences of HcrA and HcrB revealed that this latter
250 protein has an N-terminal region highly similar to HcrA; approximately, the first, N-
251 terminal 200 amino acid sequence region is 55 % identical to HcrA (Fig. S4A). Moreover,
252 both genes are expressed in a single transcript with a 2 base pair intergenic region. Given

253 these observations, the co-expression and purification of HcrA plus HcrB-His6 could result
254 in the purification of the active holoenzyme.
255 Size exclusion chromatography was used to determine the oligomeric state of the
256 individually purified HcrA, HcrB, and HcrAB protein forms. The elution profile of HcrA
257 displayed a single, symmetric peak that is consistent with a molecular species with an
258 average mass in solution of 44 kDa. Since the molecular mass expected for HcrA is 22,9
259 kDa this result indicates that the most probable species in solution for HcrA is a dimer.
260 Unfortunately, the results obtained with HcrB or HcrAB samples were not conclusive since
261 numerous peaks corresponding to high molecular mass species were observed, indicating
262 protein aggregation. Nonetheless, the smallest species observed with freshly prepared
263 samples were close to a homodimeric complex for HcrB (170 kDa) or a heterodimeric
264 complex for HcrAB (110 kDa) (data not shown).
265 In addition to the HcrB aggregation in solution observed during size exclusion
266 chromatography, it was observed that this protein became partially hydrolyzed after its
267 purification with the end product of this partial hydrolysis being a well-defined species with
268 an approximate molecular mass of 57 kDa according to SDS-PAGE (Fig. 7B). It is to note
269 that when HcrB was co-produced with HcrA, this proteolysis was significantly avoided. To
270 identify the 57 kDa molecular species, the corresponding gel band was subjected to
271 fingerprint mass spectrometry. This analysis revealed that this core and stable fragment
272 corresponded to the C-terminal 523 amino acid residues of HcrB (56,906 Da molecular
12
273 mass) and therefore the hydrolysis occurred between the K289 and Q290 residues. Hence,
274 QVAQPAV is the N-terminal sequence of this fragment.
275 A domain analysis of the HcrB protein at the NCBI site revealed the presence of
276 three conserved domains (Fig. S4B). From the N- to C-terminal of the protein sequence, it

277 could be found the COG0431 (1 to 175 amino acid residue, NAD(P)H-dependent FMN
278 reductase, SsuE), COG3976 (179 to 275, FMN-binding domain, FMN_bind), and
279 COG1053 (321 to 797, succinate dehydrogenase/fumarate reductase, flavoprotein subunit,
280 SdhA) domains. As determined by the proteomic analysis, the N-terminal end of the 57
281 kDa fragment would be located between the domains involved in FMN-reductase activity
282 (COG0431 and COG3976) and the FAD binding domain (COG1053). The proposed model
283 for the modular tridimensional structure of HcrB protein bound to FMN and FAD is shown
284 in Fig. S4C.
285 The protein domain analysis of the HcrA protein showed the presence of only the
286 COG0431 domain. Therefore, the COG0431 domain is present in the two contiguous
287 proteins, HcrA and HcrB. The presence of two similar genes predicted to encode two 20
288 kDa proteins each containing flavin mononucleotide (FMN) reductase conserved domains
289 has been previously described in Lactobacillus johnsonii and other lactobacilli (16, 17).
290 Similarly to L. plantarum HcrAB reductase, in the L. johnsonii reductase, the co-expression
291 of both reductase proteins facilitated the solubility of the active heterodimer. The genes
292 encoding the subunits of this reductase are conserved in all other members of the L.
293 acidophilus group (L. gasseri, L. acidophilus, and L. delbrueckii). Homology searches in
294 more distant species revealed that genes homologous to those from L. plantarum and L.
295 johnsonii are encountered as consecutive genes in several species belonging to the
296 Streptococcus, Enterococcus, and Pediococcus genera. In all these cases, the first, upstream
13
297 gene is similar in sequence and size to HcrA (or lj_0548 and lj_0549 from L. johnsonii),
298 and is followed by a second, larger gene, of which the N-terminal, 200 amino acid residue
299 domain is homologous to the HcrB N-terminal domain. This type of arrangement is also
300 present in a more distant species from the Actinobacteriaceae class, and Atopobium

301 parvulum (16). In all the aforementioned species, the proteins encoded by these genes are
302 annotated as fumarate reductases, NADH dehydrogenases, or flavin reductases. Apart from
303 the L. johnsonii reductase, there are no experimental data supporting these annotations. The
304 only proteins described so far for hydroxycinnamate reductase activity are enoate
305 reductase; however, this work on the HcrA and HcrB proteins in L. plantarum describes a
306 new type of heterodimeric enzymes involved in the reduction of the carbon-carbon double
307 bond present in hydroxycinnamic acids.
308 Hydroxycinnamate reduction in lactic acid bacteria. Once the involvement of L.
309 plantarum hcr genes in hydroxycinnamate reductase activity was demonstrated, it seems
310 probable that other species of lactic acid bacteria could also reduce these phenolic acids.
311 Database searches revealed the existence of similar hcr genes in other lactic acid bacteria
312 species (Fig. 8). The conservation of the same hcrAB genetic organization in these bacteria
313 supports the involvement of HcrA protein in hydroxycinnamate reduction. The amino acid
314 sequences of HcrA and HcrB homologues from eleven lactic acid bacterial species were
315 aligned (Fig. S5 and S6, respectively). The degree of identity exhibited among these HcrA-
316 like proteins ranged from 50% (Enterococcus columbae and Streptococcus mutans
317 proteins) to 99% (E. casseliflavus and E. flavescens proteins). The identity among HcrB
318 proteins was slightly higher, ranging from 63% (Lactobacillus pentosus and Clostridium
319 beijerinckii) to 99% (E. casseliflavus and E. flavescens). These alignments allowed the
320 identification of conserved amino acids domains. Taking into account the high identity
14
321 found among HrcA and HcrB proteins (50%), only domains from HcrB were chosen to
322 design degenerate oligonucleotides to detect its presence by PCR. Oligonucleotides 1655
323 and 1656 amplified a 1.4-kb internal fragment of hcrB in lactic acid bacteria.
324 In order to associate the presence of the hcrB gene and the ability to reduce

325 hydroxycinnamic acids, selected strains of lactic acid bacteria were grown in culture
326 medium containing m-coumaric acid, and their supernatants were analysed for the
327 production of 3-HPPA (Fig. 9). In addition, their DNAs were used as templates in PCRs
328 experiments using oligonucleotides 1655 and 1656 to detect the presence of the hcrB gene
329 (Fig 9A). The strains belonging to L. brevis, L. sakei, L. fructivorans, Leuconostoc citreum,
330 E. durans, E. faecalis, E. faecium, and E. hirae did not amplify the expected fragment.
331 Contrarily, PCR experiments gave a positive amplicon in all the strains belonging to the L.
332 plantarum group analysed (L. plantarum, L. pentosus, and L. paraplantarum), and in E.
333 casseliflavus DSM 20680, E. gallinarum DSM 24841, and S. gallolyticus UCN34 strains.
334 These results indicated that hcrB is a gene conserved in all the strains from the L.
335 plantarum group. Database searches in complete genomes from these strains, confirmed
336 this result. Surprisingly, differences were found among the presence of hcrB among
337 enterococcal strains. HcrB-like proteins were found in E. casseliflavus and E. gallinarum
338 species. However, in spite that numerous E. faecium and E. faecalis strains have been
339 sequenced, proteins similar to HcrB were only found in E. faecalis 06-MB-DW-09 strain
340 (S4D3PU_ENTFL accession) and two E. faecium strains (T2NMF6_ENTFC and
341 A0A24J1P6_ENTFC accessions). Strain variability and the possibility of an erroneous
342 taxonomic identification of these strains could not be discarded (18).
343 HPLC analysis of the supernatants from cultures of these bacteria in the presence of
344 m-coumaric acid indicated that only those possessing the hcrB gene were able to reduce this
15
345 hydroxycinnamic acid to 3-HPPA (Fig 9B). Only strains from the L. plantarum group as
346 well as E. casseliflavus DSM 20680, E. gallinarum DSM 24841, and S. gallolyticus
347 UCN34 strains, produced 3-HPPA from m-coumaric acid. However, the strains analysed
348 from the L. brevis, L. sakei, L. fructivorans, Leuconostoc citreum, E. durans, E. faecalis, E.

349 faecium, and E. hirae species did not reduce the hydroxycinnamic acid present in the
350 culture media. From these bacterial species, only in L. brevis the metabolism of
351 hydroxycinnamates have been studied (19), L. brevis strains were not able to reduce the
352 hydroxycinnamic acids assayed.
353 The results obtained suggest that the ability to reduce hydroxycinnamates is not
354 widely extended among lactic acid bacterial strains. Moreover, the ability to reduce these
355 acids is related to the presence of the hcrB gene found in these bacteria. Hydroxycinnamate
356 reductase activity has not been widely studied in lactic acid bacteria. In addition to L.
357 plantarum, the ability to reduce some hydroxycinnamic acids has been only described in L.
358 paracollinoides (formerly L. pastorianus var. quinicus (20, 21), L. fermentum (22, 23), L.
359 curvatus (24), L. reuteri (23), L. rossiae (24), Weisella cibaria (24), Clostridium
360 aerotolerans (25), and Clostridium xylanolyticum (25) strains. As these two latter
361 clostridial species, in addition to hydroxycinnamates, also reduced cinnamic acid and other
362 cinnamates (o-, m- and p-methoxycinnamic acid, p-methylcinnamic, and 3,4,5-
363 trimethoxycinnamic acid) the presence of an enoate reductase in these strains could be
364 envisaged.
365 In this work, the description of the L. plantarum heterodimeric NADH-dependent
366 hydroxycinnamate reductase identifies the alternative metabolism to decarboxylation of
367 hydroxycinnamates in lactic acid bacteria. Barthelmebs et al. (2000) (3) demonstrated that
368 L. plantarum has three inducible enzymatic activities for the degradation of
16
369 hydroxycinnamates involved in the stress response induced by phenolic acids particularly
370 in acidic media, which are the natural habitats of lactic acid bacteria (Fig. 10). PDC
371 decarboxylase production appears as the most efficient and rapid cellular response to
372 convert these acids into less toxic derivatives (3). The physiological significance of the

373 hydroxycinnamate metabolism has been poorly investigated but its degradation is a
374 mechanism to detoxify such compounds. The reduction of hydroxycinnamic acids involves
375 a hydrogen donor and the reoxidation of the reduced cofactor NADH, thus providing a
376 metabolic advantage through NAD+ regeneration. It has been suggested that strictly
377 heterofermentative lactic acid bacteria mainly use hydroxycinnamic acids as external
378 acceptors of electrons and gain additional metabolic energy to counteract the stressful
379 conditions generated by phenolics (24).
380 So far, only enoate reductases have been described for the reduction of
381 cinnamates/hydroxycinnamates. However, this work identifies the L. plantarum
382 hydroxycinnamate reductase and provides a genetic description of a 2-coumarate reductase
383 (subgroup EC 1.3.1.11) from the oxidorreductases of the class EC 1.3.1 acting on carbon-
384 carbon double bond.
385
386 MATERIALS AND METHODS
387
388 Bacterial strains and growth conditions. L. plantarum WCFS1 used through this study
389 was kindly provided by Dr. Michiel Kleerebezem (NIZO Food Research, The Netherlands).
390 This strain is a colony isolate of L. plantarum NCIMB 8826, which was isolated from
391 human saliva. In this study, additional 25 L. plantarum strains were analyzed. L. plantarum
392 NC8 was kindly provided by L. Axelsson (Norwegian Institute of Food, Fisheries and
17
393 Aquaculture Research, Norway). Seven strains were purchased from the Spanish Type
394 Culture Collection (CECT): L. plantarum CECT 220 (ATCC 8014), CECT 221 (ATCC
395 14431), CECT 223, CECT 224, CECT 749 (ATCC 10241), CECT 4645 (NCFB 1193), and
396 the type strain L. plantarum subsp. plantarum CECT 748T (ATCC 14917, DSMZ 20174).

397 Six strains were purchased from the German Collection of Microorganisms and Cell
398 Cultures (DSMZ): L. plantarum DSM 1055, DSM 2648, DSM 10492, DSM 13273, DSM
399 20246, and the type strain of L. plantarum subsp. argentoratensis DSM 16365T. Ten strains
400 were isolated from must grape or wine of different wine-producing areas of Spain over the
401 period from 1998 to 2001: L. plantarum RM28, RM31, RM35, RM38, RM39, RM40,
402 RM41, RM71, RM72, and RM73) (26). In addition, two Lactobacillus paraplantarum and
403 three Lactobacillus pentosus strains purchased from the DSMZ, DSM 10641 (ATCC
404 10776) and DSM 10667T, and DSM 16366, DSM 20314, and DSM 20199, respectively,
405 were also analyzed. Strains from other lactic acid species were also purchased from the
406 CECT or DSMZ collections such as Enterococcus casseliflavus (DSM 20680), E. durans
407 (DSM 20633), E. faecalis (DSM 20478), E. faecium (CECT 4102, and DSM 20477), E.
408 gallinarum (DSM 24841), E. hirae (DSM 20160), Lactobacillus brevis (CECT 5354,
409 CECT 216, and CECT 4121), L. fermentum (CECT 4007), L. fructivorans (CECT 4785), L.
410 sakei (DSM 15831), and Leuconostoc citreum (CECT 4025 and CECT 4700).
411 Streptococcus gallolyticus subsp. gallolyticus UCN34 (CIP 110142) was kindly provided
412 by Dr. Philipe Glaser (Institut Pasteur, France).
413 Lactic acid bacteria were routinely grown on MRS broth. When hydroxycinnamate
414 reductase activity was assayed, MRS media was supplemented with filter-sterilized
415 hydroxycinnamic acids at 1.5 mM final concentration. Where appropriate, erythromycin
416 was added to the culture medium at 10 μg/ml and lincomycin at 100 μg/ml. The inoculated
18
417 media were incubated at 30 ºC in the dark, without shaking for 7 days. Incubated media
418 with cells and without hydroxycinnamic acid were used as controls. The phenolic
419 compounds present in the supernatants were extracted by a standard protocol involving two
420 extraction steps with one-third of the reaction mixture volume of ethyl acetate.

421 Construction of L. plantarum hcr knockout mutants. To ascertain the
422 participation of hcr genes on hydroxycinnamate reduction, insertion-duplication
423 mutagenesis was employed. Internal fragments from the hcrR (lp_1422), hcrA (lp_1424),
424 hcrB (lp_1425) and hcrC (lp_1426) genes were cloned into the suicide pUCE191 vector
425 (27) by using primers described in Table 1. When pUCE191 and its derivatives were used
426 as donor DNA, L. plantarum transformants were selected by plating with 100 μg/ml
427 lincomycin and 10 μg/ml erythromycin, and E. coli transformants by plating with
428 ampicillin at 100 μg/ml. Derivative-pUCE191 plasmids, constructed in E. coli, were used to
429 transform L. plantarum WCFS1 competent cells by electroporation according to the method
430 of Aukrust and Blom (1992) (28). Knockout mutants were selected by plating in MRS
431 plates containing erythromycin and lincomycin. The correct insertion of the donor
432 pUCE191-derivative plasmid into the L. plantarum WCFS1 chromosome was checked by
433 PCR analysis using primers flanking the target region combined with vector-specific
434 primers (Table 1).
435 RNA isolation, reverse transcription-PCR, and quantitative PCR. L. plantarum
436 WCFS1 MRS cultures were grown to an OD600 of 0.8 to 0.9 and then supplemented with p-
437 coumaric acid or other cinnamic acids at 1.5 mM final concentration. After 10 min of
438 incubation, the cultures were immediately processed for RNA extraction as previously
439 described (29). After DNaseI treatment, the DNA-free RNA was retrotranscribed using the
440 High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the
19
441 manufacturer instructions. The cDNA obtained in presence of p-coumaric acid was used for
442 RT-PCR experiments. The hcrR (lp_1422), hcrA (lp_1424), hcrB (lp_1425), hcrC
443 (lp_1426), and lp_1427 genes, as well as the hcrR-hcrA, hcrA-hcrB, hcrB-hcrC, hcrC-
444 lp_1427 intergenic regions were PCR analyzed. PCR amplifications were performed using

445 1µl of cDNA template and 0.5 µM each of the corresponding primers (Table 1) for 35
446 cycles (94 ºC for 30 s, 55 ºC for 45 s, and 72 ºC for 1 min). As a control, PCR of DNase-
447 treated RNA was performed with the same primers to check for DNA contamination.
448 Quantitative gene expression analyses were performed in an AbiPrism 7500 Fast
449 Real Time PCR system. Specific primer pairs were designed with the Primer Express 3.0
450 program to amplify gene internal regions and the endogenous control gene (ldh) (30) (Table
451 1). The SYBR Green method was used and each assay was performed in triplicate using
452 SYBR Green real-time PCR Master Mix (Applied Biosystems). Amplification was initiated
453 at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Control
454 PCRs were included to confirm the absence of primer dimer formation, and to verify that
455 there was no DNA contamination. All quantitative-PCR (qPCR) assays amplified a single
456 product, as determined by melting-curve analysis and by electrophoresis. A standard curve
457 was plotted with cycle threshold (Ct) values obtained from amplification of known
458 quantities of cDNA and used to determine the efficiency (E) as follows: E = 10-1/slope. In
459 order to measure L. plantarum gene expression, amplification of the endogenous control
460 gene was performed simultaneously, and its relative expression was compared with that of
461 the target gene.
462 Relative quantitative (RQ) expression levels were calculated with the Applied
463 Biosystems 7500 Fast System relative quantification software using the L. plantarum ldh
464 gene as the endogenous gene (30) and growth in the absence of a cinnamic acid as the
20
465 growth condition calibrator. Results were analysed using the comparative Ct method (also
466 named double delta-delta Ct (2 ΔΔCt) method).
467 Expression of hcr genes in E. coli and purification of the recombinant proteins.
468 The hcrA, hcrB, and hcrAB genes from L. plantarum WCFS1 were PCR-amplified by HS

469 Prime Start DNA polymerase (Takara) by using the primer pairs 1631-1632 (hcrA), 889-
470 890 (hcrB), and 1631-890 (hcrAB). The purified PCR products were inserted into the
471 pURI3-Cter vector following the restriction enzyme- and ligation-free cloning strategy
472 described previously (31). The vectors produce recombinant proteins having a six-histidine
473 affinity tag in their C-termini. E. coli DH10B cells were transformed; recombinant plasmids
474 were isolated and verified by DNA sequencing, and then used to transform E. coli
475 BL21(DE3) cells for expression.
476 E. coli BL21(DE3) was transformed with the recombinant pURI3-Cter-hcrA,
477 pURI3-Cter-hcrB, and pURI3-Cter-hcrAB plasmids. E. coli cells were grown in LB m
478 containing ampicillin (100 μg/ml), until they reach an optical density at 600 nm of 0.4 and
479 induced by adding IPTG (0.4 mM final concentration). After induction, the cells were
480 grown at 22 ºC during 20 h and collected by centrifugation. Cells were resuspended in
481 phosphate buffer (50 mM, pH 6.5). To prevent inclusion body formation, E. coli cells were
482 grown at 22 ºC in medium containing 1M D-sorbitol, 2.5 mM glycine betaine, and 0.25
483 mM IPTG (32). Crude extracts were prepared by French press lysis of the cell suspension
484 (three times at 1100 psi). The insoluble fraction of the lysate was removed by
485 centrifugation at 47,000g for 30 min at 4 ºC and the supernatant was filtered through a 0.2
486 µm filter. The supernatant of the HcrA, HcrB, and HcrAB proteins was analysed for
487 hydroxycinnamate reductase by adding 1.5 mM m-coumaric acid and incubating at 37 ºC
488 during 16 h.
21
489 For purification of the recombinant His6-tagged proteins, the filtered supernatants
490 were applied to a TALON® Superflow resin (Clontech) equilibrated with the buffer
491 described previously containing 0.3 M NaCl and 10 mM imidazole to improve the
492 interaction specificity in the affinity chromatography step. The bound enzymes were eluted

493 using 150 mM imidazole in the same buffer. The purity of the enzymes was determined by
494 sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in Tris-glycine
495 buffer.
496 Size exclusion chromatography. Fractions containing the enzymes eluted from the
497 TALON® Superflow resin (Clontech) were pooled and loaded onto a HiLoad 16/60
498 Superdex 200 prep grade column (GE Healthcare) preequilibrated in Tris buffer (20 mM
499 Tris-HCl, pH 8.5, with 0.1 M NaCl) using an ÄKTAprime plus chromatographic system
500 (GE Healthcare). Fractions with the protein of interest identified by SDS-PAGE were
501 pooled and the protein was concentrated and stored at −80 °C until its use. Protein
502 concentration was determined spectrophotometrically by UV absorption using extinction
503 coefficients estimated with the ExPASy server (https://web.expasy.org/protparam/).
504 Enzyme activity assay. Fractions containing the His6-tagged proteins were pooled
505 and analyzed for hydroxycinnamate reductase activity. Eluted purified HcrA, HcrB, and
506 HcrAB proteins (500 μg) were incubated at 37 ºC during 18 h in the presence of 1.5 mM m-
507 coumaric acid and 15 mM NAD(P)H, 7.5 mM FMN, and 7.5 mM FAD. The phenolic
508 compounds present in the reaction were extracted by ethyl acetate and analysed by HPLC
509 with diode array detection (DAD) as described previously (5).
510 PCR detection of hydroxycinnamate reductase activity. Genes encoding HcrB
511 proteins were amplified by PCR using chromosomal DNA from several lactic acid bacterial
512 strains. Amplifications were performed by using degenerate primers 1655 and 1656. The
22
513 reactions were performed using 30 cycles of denaturation at 94 ºC for 30 s, annealing at 55
514 ºC for 1 min, and extension at 72 ºC for 30 s. The expected size of the amplicon was 1.4 kb.
515 Protein identification via MS. The protein band was excised manually and then
516 digested automatically using a Proteineer DP protein digestion station (Bruker-Daltonics,

517 Bremen, Germany). The protocol described by Shevchenko et al. (1996) (33) was used for
518 trypsin digestion. The digestion was analysed in an Ultraflex time-of-flight mass
519 spectrometer (MS; Bruker-Daltonics) equipped with a LIFT-MS/MS device. Spectra were
520 acquired in the positive-ion mode at a 50-Hz laser frequency, and 100 to 1000 individual
521 spectra were averaged. Automated analysis of mass data was performed using flexAnalysis
522 software (Bruker-Daltonics). Matrix-assisted laser desorption ionization MS and MS/MS
523 data were combined through the BioTools program (Bruker-Daltonics) to search a
524 nonredundant protein database (NCBInr Swiss-Prot) using Mascot software (Matrix
525 Science, London, United Kingdom).
526 Sequence data analyses. A homology search with finished and unfinished
527 microbial genome databases was performed with the BLAST algorithm at the National
528 Center for Biotechnology Information served
529 (http://www.ncbi.nim.nih.gov/sutils/genom_table.cgi). Multiple alignments were made
530 using the Clustal Omega program, (http://www.ebi.ac.uk/Tools/msa/clustalo/) on the EBI
531 site, after retrieval of sequences from the GenBank and Swiss-Prot databases. Computer
532 promoter predictions were carried out at the Internet site
533 (http://www.friutfly.org/seq_tools/promoter.html), and predicted transcription terminators
534 were analysed at the RNAold site
535 (http://ran.igmors.u_psud.fr/tool/box/arnold/index.php#Results).
23
536 Statistical analysis. The two-tailed Students t test performed using GraphPad InStat
537 version 3.0 (GrapPad Software, San Diego, CA) was used to determine the differences
538 between means. The data are representative means of at least three independent
539 experiments.

540
541
542 ACKNOWLEDGEMENTS
543 We are grateful to J. M. Mancheño for the exclusion size chromatography and the critical
544 reading of the manuscript. M. V. Santamaría and J. M. Barcenilla for their assistance. L.
545 Santamaría is a recipient of a FPI Fellowship from the MINEICO.
546
547
548 FUNDING INFORMATION
549 This work was supported by grant AGL2014-52911-R (AEI/FEDER, UE) (MINEICO).
550
551
552 REFERENCES
553 1. Shahidi F, Naczk M. 2003. Phenolics in food and nutraceuticals. CRC Press, London,
554 United Kingdom.
555 2. Rodríguez H, Curiel JA, Landete JM, de las Rivas B, López de Felipe F, Gómez-
556 Cordovés C, Mancheño JM, Muñoz R. 2009. Food phenolics and lactic acid bacteria.
557 Int J Food Microbiol 132:79-90. https://doi.org/10.1016/j.ijfoodmicro.2009.03.025
558 3. Barthelmebs L, Diviès C, Cavin J-F. 2000. Knockout of the p-coumarate decarboxylase
559 gene from Lactobacillus plantarum reveals the existence of two other inducible
24
560 enzymatic activities involved in phenolic acid metabolism. Appl Environ Microbiol
561 66:3368-3375.
562 4. Cavin JF, Barthelmebs L, Guzzo J, van Breeumen J, Samyn R, Travers JF, Diviès C,
563 1997. Purification and characterization of an inducible p-coumaric acid decarboxylase

564 from Lactobacillus plantarum. FEMS Microbiol Lett 147:291-295.
565 https://doi.org/10.1016/S0378-1097(97)00004-9
566 5. Rodríguez H, Landete JM, Curiel, JA, de las Rivas B, Mancheño JM, Muñoz R. 2008.
567 Characterization of the p-coumaric acid decarboxylase from Lactobacillus plantarum
568 CECT 748T. J Agric Food Chem 56:3068-3072. doi: 10.1021/jf703779s
569 6. Rodríguez H, Landete JM, de las Rivas B, Muñoz R. 2008. Metabolism of food
570 phenolic acids by Lactobacillus plantarum CECT 748T. Food Chem 107:1393-1398.
571 https://doi.org/10.1016/j.foodchem.2007.09.067
572 7. Reverón I, de las Rivas B, Muñoz R, López de Felipe F. 2012. Genome-wide
573 transcriptomic responses of a human isolate of Lactobacillus plantarum exposed to p-
574 coumaric acid stress. Mol Nutr Food Res 56:1848-1859. doi: 10.1002/mnfr.201200384
575 8. Sun J, Lin Y, Shen X, Jain R, Sun X, Yuan Q, Yan Y. 2016. Aerobic biosynthesis of
576 hydrocinnamic acids in Escherichia coli with a strictly oxygen-sensitive enoate
577 reductase. Met Engineer 35:75-82. doi: 10.1016/j.ymben.2016.02.002
578 9. Levy CC. 1964. Metabolism of coumarin by a microorganism: o-coumaric acid as an
579 intermediate between coumarin and melilotic acid. Nature 4963:1059-1061.
580 10. Levy CC, Weinstein GD. 1964. The metabolism of coumarin by a microorganism. II.
581 The reduction of o-coumaric acid to melilotic acid. Biochem 3:1944-1947. DOI:
582 10.1021/bi00900a027
25
583 11. Caldeira J, Feicht R, White H, Teixeira M, Moura JJG, Simon H, Moura I. 1996. EPR
584 and Mössbauer spectroscopic studies on enoate reductases. J Biol Chem 271:18743-
585 18748. doi: 10.1074/jbc.271.31.18743
586 12. Rohdich F, Wiese A, Feicht R, Simon H, Bacher A. 2001. Enoate reductases of

587 Clostridia. J Biol Chem 276:5779-5787. doi: 10.1074/jbc.M008656200
588 13. Bühler M, Simon H. 1982. On the kinetics and mechanism of enoate reductase. Hoppe-
589 Seyler´s Z Physiol Chem 363:609-625.
590 14. Bougioukou DJ, Stewart JD. 2012. Reduction of C=C double bonds. Enzyme catalysis
591 in Organic Synthesis. Third Edition. Ch. 27, 1111-1163, Wiley-VCH Verlag
592 15. Mordaka PM, Hall SJ, Minton N, Stephens G. 2018. Recombinant expression and
593 characterisation of the oxygen-sensitive 2-enoate reductase from Clostridium
594 sporogenes. Microbiology 164:122-132. doi: 10.1099/mic.0.000568. doi:
595 10.1099/mic.0.000568
596 16. Hertzberger R, Arents J, Dekker HL, Pridmore RD, Gysler C, Kleerebezem M, Teixeira
597 de Mattos M. J. 2014. H2O2 production in species of the Lactobacillus acidophilus
598 group: a central role for a novel NADH-dependent flavin reductase. App Environ
599 Microbiol 80:2229-2239. doi: 10.1128/AEM.04272-1
600 17. Valladares RB, Graves C, Wright K, Gardner CL, Lorca GL, González CF. 2015. H2O2
601 production rate in Lactobacillus jonhsonii is modulated via the interplay of a
602 heterodimeric flavin oxidoreductase with a soluble 28 kd PAS domain containing
603 protein. Front Microbiol 6:716. doi: 10.3389/fmicb.2015.00716
604 18. Papadimitriou-Olivgeris M, Filippidou S, Kolonitsiou F, Drougka E, Koutsileou K,
605 Fligou F, Dodou V, Sarrou S, Marangos M, Vantarakis A, Anastassiou ED, Petinaki E,
606 Spiliopoulou I. 2016. Pitfalls in the identification of Enterococcus species and the
26
607 detection of vanA and vanB genes. Lett Appl Microbiol 63:189-195. doi:
608 10.1111/lam.12610
609 19. Curiel JA, Rodríguez H, Landete JM, de las Rivas B, Muñoz R. 2010. Ability of
610 Lactobacillus brevis strains to degrade food phenolic acids. Food Chem 120:225-229.

611 https://doi.org/10.1016/j.foodchem.2009.10.012
612 20. Ehrmann MA, Vogel RF. 2005. Taxonomic note “Lactobacillus pastorianus” (Van
613 Laer, 1892) a former synonym for Lactobacillus paracollinoides. Syst Appl Microbiol
614 28:54-56. DOI:10.1016/j.syapm.2004.09.007
615 21. Whitting GC, Carr JG. 1959. Metabolism of cinnamic acid and hydroxycinnamic acids
616 by Lactobacillus pastorianus var. quinicus. Nature 4696:1427-1428. DOI:
617 10.1038/1841427a0
618 22. Sánchez-Maldonado AF, Schieber A, Gänzle MG. 2011. Structure-function
619 relationships of the antibacterial activity of phenolic acids and their metabolism by
620 lactic acid bacteria. J. Appl. Microbiol. 111:1176-1184. doi: 10.1111/j.1365-
621 2672.2011.05141.x
622 23. Filannino P, Bai Y, di Cagno R, Gobbetti M, Gänzle MG. 2015. Metabolism of
623 phenolic compounds by Lactobacillus spp. During fermentation of cherry juice and
624 broccoli puree. Food Microbiol. 46:272-279. doi: 10.1016/j.fm.2014.08.018
625 24. Filanino P, Gobbetti M, de Angelis M, di Cagno R. 2014. Hydroxycinnamic acids used
626 as external acceptors of electrons: an energetic advantage for strictly heterofermentative
627 lactic acid bacteria. Appl. Environ. Microbiol. 80:7574-7582. doi:
628 10.1128/AEM.02413-14
629 25. Chamkha M, Garcia JL, Labat M. 2001. Metabolism of cinnamic acids by some
630 Clostridiales and emmendation of the descriptions of Clostridium aerotolerans,
27
631 Clostridium celerecrescens and Clostridium xylanolyticum. Int. J. Syst. Evol.
632 Microbiol. 51:2105-2111. DOI:10.1099/00207713-51-6-2105
633 26. Moreno-Arribas MV, Polo MC, Jorganes F, Muñoz R. 2003. Screening of biogenic
634 amine production by lactic acid bacteria isolated from grape must and wine. Int J Food

635 Microbiol 84:117-123.
636 27. Arrecubieta C, García E, López R. 1995. Sequence and transcriptional analysis of a
637 DNA region involved in the production of capsular polysaccharide in Streptococcus
638 pneumoniae type 3. Gene 167:1-7.
639 28. Aukrust T, Blom H. 1992. Transformation of Lactobacillus strains used in meat and
640 vegetable fermentations. Food Res Int 25:253-261. https://doi.org/10.1016/0963-
641 9969(92)90121-K
642 29. Saulnier DM, Molenaar D, de Vos WM, Gibson GR, Kolida S. 2007. Identification of
643 prebiotic fructooligosaccharide metabolism in Lactobacillus plantarum WCFS1
644 through microarrays. Appl Environ Microbiol 73:1753-1765. doi:10.1128/AEM.01151-
645 06
646 30. Fiocco D, Crisetti E, Capozzi V, Spano G. 2008. Validation of an internal control gene
647 to apply reverse transcription quantitative PCR to study heat, cold and ethanol stresses
648 in Lactobacillus plantarum. World J Microbiol Biotechnol 24:899-902. DOI:
649 10.1007/s11274-007-9556-7
650 31. Curiel JA, de las Rivas B, Mancheño JM, Muñoz R. 2011. The pURI family of
651 expression vectors: a versatile set of ligation independent cloning plasmids for
652 producing recombinant His-fusion proteins. Prot Expr Purif 76:44-53. doi:
653 10.1016/j.pep.2010.10.013
28
654 32. Ackerley DF, González CF, Park CH, Blake R, Keyhan M, Martin A. 2004. Chromate-
655 reducing properties of soluble flavoproteins from Pseudomonas putida and Escherichia
656 coli. Appl Environ Microbiol 70:873-882. doi: 10.1128/AEM.70.2.873-882.2004
657 33. Shevchenko A, Wilm M, Vorm O, Mann M. 1996. Mass spectrometric sequencing of

658 proteins from silver-stained polyacrylamide gels. Anal Chem 68:850-858.
659
660
661 Legends to Figures
662
663 FIG 1 Effect of disruption of lp_1424 (hcrA) and lp_1425 (hcrB) on hydroxycinnamate
664 reduction in L. plantarum WCFS1. HPLC chromatograms of L. plantarum cultures
665 incubated in 1.5 mM m-coumaric acid are shown for L. plantarum WCFS1 (wild type [wt]),
666 L. plantarum WCFS1 (pUCE191-hcrA) (Δlp_1424 mutant), and L. plantarum WCFS1
667 (pUCE191-hcrB) (Δlp_1425 mutant). Results for uninoculated medium are also shown
668 (control). The m-coumaric acid (mCA) and 3-(3-hydroxyphenyl) propionic acid (3-HPPA)
669 detected arte indicated. Chromatograms were recorded at 280 nm.
670
671 FIG 2 Genetic organization of the L. plantarum WCFS1 chromosomal region containing
672 the hydroxycinnamate reductase encoding genes. (A) (accession NC_004567, positions
673 1299251-1309330). Arrows indicate genes. The shaded genes encode genes putatively
674 involved in hydroxycinamate reduction (hcr). The location of putative promoters and
675 transcription terminators are also indicated. The size and direction of the transcripts
676 revealed by reverse transcription is also shown. (B) Transcriptional analysis by RT-PCR of
677 the L. plantarum WCFS1 genome in hydroxycinnamate reductase locus. RT-PCR
29
678 amplification with primers designed to amplify internal gene regions or intergenic regions:
679 hcrR (primers 1328+1329, 628 bp) (2), hcrR-hcrA (1327+1229, 861 bp) (3), hcrA
680 (1228+1229, 241 bp) (4), hcrA-hcrB (1222+1330, 432 bp) (5), hcrB (877+878, 758bp) (6),
681 hcrB-hcrC (1385+1052, 778 bp) (7), hcrC (1051+1952, 384 bp) (8), hcrC-lp_1427

682 (1226+1331, 323 bp) (9), and lp_1427 (1332+1333, 395 bp) (10). The control without
683 cDNA is also shown (1). Left lane, 100-bp molecular size ladder. Numbers indicate some
684 of the molecular sizes.
685
686 FIG 3 Relative expression levels of hcrAB genes on hcr knockout mutants in response to
687 1.5 mM m-coumaric acid. Expression levels were calculated with the 7500 Fast System
688 relative quantification software using L. plantarum ldh gene as endogenous gene and the
689 growth in the absence of m-coumaric acid as growth condition calibrator. Expression level
690 of hcrA and hcrB genes are represented by black and grey bars, respectively. The
691 experiments were done in triplicate. The mean value and the standard error are shown.
692 Asteriks indicate a p value <0.1.
693
694 FIG 4 Purification and enzymatic activity of E. coli extracts expressing L. plantarum HcrB
695 and recombinant HcrB protein. (A) SDS-PAGE analysis of the expression and purification
696 of the His6-tagged HcrB. Results of analysis of soluble cell extracts of IPTG-induced E.
697 coli BL21(DE3) (pURI3-Cter) (lane 1) or E. coli BL21(DE3) (pURI3-Cter-HcrB) (lane 2),
698 flowthrough from the affinity resin (lane 3), or fractions eluted after His affinity resin
699 (lanes 4 to 8). The 8% gel was stained with Coomassie blue. Molecular mass markers are
700 located on the left (SDS-PAGE standards; BioRad). (B) HPLC chromatograms showing
701 hydroxycinnamate reductase activity of soluble cell extracts of IPTG-induced E. coli
30
702 BL21(DE3) (pURI3-Cter) (control) or E. coli BL21(DE3) (pURI3-Cter-HcrB) (HcrB)
703 incubated in 1.5 mM m-coumaric acid. HPLC chromatograms also showed the reductase
704 activity of purified His6-HcrB protein (500 µg) (HcrB) or without protein (control). The m-
705 coumaric acid (mCA) and 3-(3-hydroxyphenyl) propionic acid (3-HPPA) detected are

706 indicated. Chromatograms were recorded at 280 nm.
707
708 FIG 5 Reductase activity of HcrB on several hydroxycinnamic acids. HPLC
709 chromatograms of E. coli BL21(DE3) (pURI3-Cter) (control) or E. coli BL21(DE3)
710 (pURI3-Cter-hcrB) (HcrB) cell free extracts incubated at 37 ºC during 16 h. The
711 hydroxycinnamic acids assayed were p-coumaric (pCA), m-coumaric (mCA), o-coumaric
712 (oCA), ferulic acid (FA), caffeic acid (CA), and sinapic acid (SA). A non-hydroxy derived
713 cinnamic acid was used as control (cinnamic acid, CiA). The corresponding reduced acids,
714 such as phloretic acid (PhlA), 3-(3-hydroxyphenyl) propionic (3-HPPA), 3-(2-
715 hydroxyphenyl) propionic (2-HPPA) or melilotic, hydroferulic (HFA), and hydrocaffeic
716 (HCA), or hydrosinapic acid (HAS) detected are indicated. Chromatograms were recorded
717 at 280 nm.
718
719 FIG 6 Relative transcriptional expression of hcr genes in L. plantarum WCFS1 in response
720 to the presence of several hydroxycinnamic acids. L. plantarum cultures were exposed
721 during 10 min at 1.5 mM of a hydroxycinnamic acid (p-coumaric, m-coumaric, o-coumaric,
722 ferulic, caffeic, or sinapic acid). As controls, a non-hydroxy derived cinnamic acid
723 (cinnamic acid) and a hydroxybenzoic acid (gallic acid) were assayed. Expression levels
724 were calculated with the 7500 Fast System relative quantification software using L.
725 plantarum ldh gene as endogenous gene and the growth in the absence of acid as growth
31
726 condition calibrator. Expression level of hcrR, hcrA, hcrB and hcrC genes are represented
727 by brick-like shading, black colour, grey colour, and dotted-like shading bars, respectively.
728 The experiments were done in triplicate. The mean value and the standard error are shown.
729 Asteriks indicate a p value <0.1.

730
731 FIG 7 SDS-PAGE analysis of the production and purification of HcrA, HcrB, and HcrAB
732 recombinant proteins. (A) Soluble cell extracts of IPTG-induced E. coli BL21(DE3)
733 (pURI3-Cter) (lane 1), E. coli BL21(DE3) (pURI3-Cter-HcrA) (lane 2), E. coli BL21(DE3)
734 (pURI3-Cter-HcrB) (lane 3), and E. coli BL21(DE3) (pURI3-Cter-HcrAB) (lane 4). (B)
735 Purification of HrcA (lane 2), HcrB (lane 4) and HcrAB (lane 6) recombinant proteins from
736 the corresponding E. coli BL21cell extracts [pURI3-Cter-HcrA (lane 1), pURI3-Cter-HcrB
737 (lane 3), and pURI3-Cter-HcrAB (lane 5)]. HcrA protein (22 kDa), HcrB (87 kDa), and
738 hydrolyzed-HcrB (57 kDa).
739
740 FIG 8 Genetic organization of hydroxycinnamate reductases from several lactic acid
741 bacteria, such as Lactobacillus plantarum WCFS1 (NC_004567.2), Lactobacillus
742 paraplantarum DSM 10667 (NZ_CP013130.1), Lactobacillus pentosus DSM 20314
743 (GCA_001433755.1), Lactobacillus fabifermentans DSM 21115 (NZ_AYGX00000000.2),
744 Lactobacillus alimentarius DSM 20249 (NZ_AZDQ00000000.1), Lactobacillus
745 paralimentarius DSM 13961 (NZ_AZDH00000000.1), Lactobacillus dextrinicus DSM
746 20335 (NZ_AYYK00000000.1), Enterococcus phoeniculicola ATCC BAA-412
747 (NZ_ASWE00000000.1), Enterococcus columbae DSM 7374 (NZ_AHYW00000000.1),
748 Enterococcus flavescens ATCC 49996 (NC_020995.1), Enterococcus casseliflavus ATCC
749 12755 (GCA_000191365.1), Enterococcus gilvus ATCC BAA-350
32
750 (NZ_ASWH00000000.1), Streptococcus parasanguinis ATCC 15912 (NC_015678.1),
751 Streptococcus uberis ATCC BAA-854 (NC_012004.1), Streptococcus equinus ATCC 9812
752 (NZ_JNLO00000000.1), Streptococcus gallolyticus ATCC 43143 (NZ_CP018822.1),
753 Streptococcus mutans ATCC 700610 (NC_004350.2), Clostridium beijerinckii ATCC

754 51743 (NZ_CP010086.2), and Clostridium saccharobutylicum DSM 13864
755 (NC_022571.1). Arrows indicate genes. Genes having putative identical functions are
756 represented by identical shading. The genes having brick-like shading encode putative
757 transcriptional regulators. Genes coding for putative reductases similar to HcrA and HcrB
758 are represented by black and grey arrows, respectively.
759
760 FIG 9 Hydroxycinammate reductase activity in lactic acid bacteria. (A) PCR amplification
761 of the HcrB gene. Chromosomal DNA from several lactic acid bacteria was used for PCR
762 amplification with oligonucleotides 1655 and 1656 to amplify 1.4 kb of the hcrB gene. PCR
763 products were subjected to gel electrophoresis and stained with Gel Red. Left lane,
764 λ/EcoT14I (Takara) molecular size marker. Numbers indicate some of the molecular sizes.
765 (B) HPLC chromatograms of supernatants from lactic acid bacteria grown during 10 days
766 at 30 ºC in MRS media supplemented with 1.5 mM m-coumaric acid. The m-coumaric acid
767 (mCA) and 3-(3-hydroxyphenyl) propionic acid (3-HPPA) detected are indicated.
768 Chromatograms were recorded at 280 nm. The strains assayed were Enterococcus
769 casseliflavus DSM 20680 (1), E. durans DSM 20633 (2), E. faecalis DSM 20478 (3), E.
770 faecium CECT 4102 (4), E. gallinarum DSM 24841 (5), E. hirae DSM 20160 (6),
771 Lactobacillus brevis CECT 5354 (7), L. fermentum CECT 4007 (8), L. fructivorans CECT
772 4785 (9), L. paraplantarum DSM 10641 (10), L. plantarum subsp. argentoratensis DSM
773 16365 (11), L. plantarum subsp. plantarum ATCC 14917 (CECT 748) (12), L. plantarum
33
774 DSM 10492 (13), L. pentosus DSM 16366 (14), L. sakei subsp. carnosus DSM 15831 (15),
775 Leuconostoc citreum CECT 4025 (16), and Streptococcus gallolyticus UCN34 (17).
776
777 FIG 10 Schematic representation of hydroxycinnamic acid metabolism in L. plantarum

778 WCFS1. When R1 (OH) and R2 (H), the represented compounds are p-coumaric acid (A),
779 vinyl phenol (B), and phloretic acid (C). When R1 (OH) and R2 (OH), caffeic acid (A),
780 vinyl catechol (B), and hydrocaffeic acid (C). When R1 (OH) and R2 (OCH3), the
781 compounds are ferulic acid (A), vinyl guaiacol (B), and hydroferulic acid (C). When R1 (H)
782 and R2 (OH), only the reduction reaction is carried out and the represented compounds are
783 m-coumaric acid (A), and 3-(3-hydroxyphenyl) propionic acid (C).
34
Table 1. Primers used in this study
Primers Sequence (5´→3´)* *Amplified fragment/cloning strategy

877 GGGGTACCTGCTGTCAAAGAAGTGACG *The 758-bp PCR hcrB internal fragment was double-digested with KpnI/XbaI
878 GCTCTAGAAGCTAAAATCACCGCATCAGC and cloned into KpnI/XbaI double-digested pUCE191 plasmid generating
pUCE191-hcrB
*Amplification of a 758-bp hcrB internal region
889 TAACTTTAAGAAGGAGATATACATATGAAATTTGTTGGAATTGTTGGAA *The 2.4 kb hcrB gene amplified with 889+890 oligos was introduced into
890 GCTATTAATGATGATGATGATGATGGGCCGTCACCGGATCAACGTGCTC pURI3-Cter vector by using a LIC strategy (the nucleotides pairing the vector
sequence are written in italics)
* The 3.1 kb hcrA+hcrB genes amplified with 1631+890 oligos were introduced
into pURI3-Cter vector by using a LIC strategy (the nucleotides pairing the vector
918 AACCGCGACAATGTTTTGATT *Amplification of a 62-bp lp_2057 (ldhD) fragment for RT-qPCR
919 TTGTGAACGGCAGTTTCAGTGT
976 ATGAAATTTGTTGGGATT *Amplification with oligo 1233 to corroborate the correct insertion of pUCE191-
hcrA into the L. plantarum WCFS1 chromosome
1051 GGGGTACCAACCAGCTTATATGA *The 384-bp PCR hcrC internal fragment was double-digested with KpnI/XbaI
1052 GCTCTAGATGAAAGTCGGTGATCCAA and cloned into KpnI/XbaI double-digested pUCE191 plasmid generating
pUCE191-hcrC
*Amplification of a 384-bp hcrC internal fragment
*Amplification with 1385+1052 oligos of a 778-bp hcrB-hcrC intergenic region
1222 CACTGGTGATGCCAAATATTGAA *Amplification of a 65-bp hcrA fragment for RT-qPCR
1223 GCCCTGATCATCAAAAGCTTGT *Amplification with 1222+1330 oligos of a 432-bp hcrA-hcrB intergenic region
1224 CGGCAGCCCTGACCAA *Amplification of a 198-bp hcrB fragment for RT-qPCR
1225 GCCGGCATCAACGTAACG
1226 TGACATCGACTGGCCCAAT *Amplification of a 56-bp hcrC fragment for RT-qPCR
1227 TGCCCTTTGTCAATGCTTCA *Amplification with 1226+1331 oligos of a 323-bp hcrC-lp_1427intergenic region
1228 GGGGTACCGAACAGCACCAGTAGC *The 241-bp PCR hcrA internal fragment was double-digested with KpnI/XbaI
1229 GCTCTAGAATCTGGCGTAAGTGC and cloned into KpnI/XbaI double-digested pUCE191 plasmid generating
pUCE191-hcrA
*Amplification of a 241-bp hcrA internal region
*Amplification with 1327+1229 oligos of a 861-bp hcrR-hcrA intergenic region
1233 AGCGGATAACAATTTCACACAGGA *24-mer Reverse Sequencing Primer (-48) for pUCE19/pUCE191
1242 ATGAAATTTGTTGGAATTGTTG *Amplification with oligo 1233 to corroborate the correct insertion of pUCE191-
1
hcrB into the L. plantarum WCFS1 chromosome
*Amplification with oligo 1233 to corroborate the correct insertion of pUCE191-
1243 ATGCAAAAACTAATGCCAGTC
hcrC into the L. plantarum WCFS1 chromosome
1327 CTTTTTCAACATTGCGGATGC *Amplification with oligo 1229 of a 861-bp hcrR-hcrA intergenic region
1328 CATTAACGCTTGGGGCAAAGG *Amplification of a 628-bp hcrR internal fragment
1329 GACTTATTATCAATATTTGACG
1330 CGTCAGCCGTCGCAATTTTAGTGG *Amplification with oligo 1222 of a 432-bp hcrA-hcrB intergenic region
1331 GACTTGCCCATCATCGCCTTGC *Amplification with oligo 1226 of a 323-bp hcrC-lp_1427intergenic region
1332 CGATAAAGTCGCCTTTAGGTAATTG *Amplification of a 395-bp lp_1427 internal fragment
1333 CAGTCCCGAACAAGTGACGCG
1385 TGCCGTGGCACCATTTTAC *Amplification with oligo1052 of a 778-bp hcrB-hcrC intergenic region
1627 TCGGCTGGTACCTATTGCCCCTA *The 335-bp PCR hcrR internal fragment was double-digested with KpnI/XbaI
1628 AACTTGGCGTCGTCTAGATGAC and cloned into KpnI/XbaI double-digested pUCE191 plasmid generating
pUCE191-hcrR
1630 ATGCCAAAATCAGATTCCAGCGA *Amplification with oligo 1233 to corroborate the correct insertion of pUCE191-
hcrR into the L. plantarum WCFS1 chromosome
1631 TAACTTTAAGAAGGAGATATACATATGAAATTTGTTGGGATTGTTGGGA *The 0.6 kb hcrA gene amplified with 1631+1632 oligos was introduced into
1632 GCTATTAATGATGATGATGATGATGTTCCTCCCCAGTCACTTGCCGCCA pURI3-Cter vector by using a LIC strategy (the nucleotides pairing the vector
*The 3.1 kb hcrA+hcrB genes amplified with 1631+890 oligos were introduced
into pURI3-Cter vector by using a LIC strategy (the nucleotides pairing the vector
1655 CARATHYTNGAYGCNCCNGGNGT * Degenerate oligonucleotides coding for the HcrB conserved motifs QILDAPGV
1656 CATCATYTGNGWRAANCCCATNCC (1655) and GMGF(S/T)QMM (1656). They are used to amplify a 1.4-kb internal
fragment of hcrB in lactic acid bacteria
*M = A or C; S = G or T; R = G or A; Y = C or T; D = A, G or T; H = A, C or T, and N = G, A, C or T.
Engineered restriction sites are underlined.
2
Figure 1
2000 2000
control

1000 mCA 1000
0 0
0 20 40 60 wt 80 200
200
Minutes
3-HPPA
100 100
mAU
0 0
0 20 40 Δlp_1424
60 80
Minutes
1000 1000
mCA
0 0
0 20 40 Δlp_1425
60 80
Minutes
1000 1000
mCA
0 0
0 20 40 60 80
Time (min)
Minutes
FIG 1 Effect of disruption of lp_1424 (hcrA) and lp_1425 (hcrB) on hydroxycinnamate

reduction in L. plantarum WCFS1. HPLC chromatograms of L. plantarum cultures
incubated in 1.5 mM m-coumaric acid are shown for L. plantarum WCFS1 (wild type
[wt]), L. plantarum WCFS1 (pUCE191-hcrA) (Δlp_1424 mutant), and L. plantarum
WCFS1 (pUCE191-hcrB) (Δlp_1425 mutant). Results for uninoculated medium are also
shown (control). The m-coumaric acid (mCA) and 3-(3-hydroxyphenyl) propionic acid
(3-HPPA) detected arte indicated. Chromatograms were recorded at 280 nm.
Figure 2

A
HcrR HcrA HcrB HcrC
● ● ● ●
lp_1420 lp_1422 lp_1424 lp_1425 lp_1426 lp_1427
B
pb 1 2 3 4 5 6 7 8 9 10
800-
500-
200-
Figure 3

30
*
Relative transciption levels
20
* *
10
0
WCFS1 WCFS1ΔhcrA WCFS1ΔhcrB
FIG 3 Relative expression levels of hcrAB genes on hcr knockout mutants in response
to 1.5 mM m-coumaric acid. Expression levels were calculated with the 7500 Fast
System relative quantification software using L. plantarum ldh gene as endogenous
gene and the growth in the absence of m-coumaric acid as growth condition calibrator.
Expression level of hcrA and hcrB genes are represented by black and grey bars,
respectively. The experiments were done in triplicate. The mean value and the standard
error are shown. Asteriks indicate a p value <0.1.
A
Figure 4
1 2 3 4 5 6 7 8
kDa
97-
66-

45-
B
E. coli cell extracts HcrB protein
control
1000 1000
1000
control 100
500 500
500 500
mCA mCA
mAU
0 0 0 0
HcrB HcrB
0 20 40 60 80
200 200
1000
Minutes
3-HPPA
100 100
3-HPPA
500
mCA
0 0 0
0 20 40 60 80 0 20 40 60 80
Minutes
Time (min) Time (min)
FIG 4 Purification and enzymatic activity of E. coli extracts expressing L. plantarum

HcrB and recombinant HcrB protein. (A) SDS-PAGE analysis of the expression and
purification of the His6-tagged HcrB. Results of analysis of soluble cell extracts of
IPTG-induced E. coli BL21(DE3) (pURI3-Cter) (lane 1) or E. coli BL21(DE3) (pURI3-
Cter-HcrB) (lane 2), flowthrough from the affinity resin (lane 3), or fractions eluted
after His affinity resin (lanes 4 to 8). The 8% gel was stained with Coomassie blue.
Molecular mass markers are located on the left (SDS-PAGE standards; BioRad). (B)
HPLC chromatograms showing hydroxycinnamate reductase activity of soluble cell
extracts of IPTG-induced E. coli BL21(DE3) (pURI3-Cter) (control) or E. coli
BL21(DE3) (pURI3-Cter-HcrB) (HcrB) incubated in 1.5 mM m-coumaric acid. HPLC
chromatograms also showed the reductase activity of purified His6-HcrB protein (500
µg) (HcrB) or without protein (control). The m-coumaric acid (mCA) and 3-(3-
hydroxyphenyl) propionic acid (3-HPPA) detected are indicated. Chromatograms were
recorded at 280 nm.
Figure 5
2000
Control 200
HrcB
A A
200 200
1000 pCA 100 PhlA 100
0 0 0 0
B 200
0 20 40 60
B80 200

500 mCA 500 3-HPPA
100 100
0 0 0 0
500 C 500
0 20 40 60
C80
2-HPPA
oCA 200 200
250 250
0 0 0 0
D 400
D
400
mAU
200 200
FA 200 HFA 200

100 100
0 0 0 0
0 20 40 60
E
80
200 200
E 200
200 HCA
CA
100 100 100 100
0 0 0 0
100
0 20 40 60
F
80
100
100
F 100
50
HSA 50
50 SA 50
0 0 0 0
G
0 20 40 60 80
1000 1000
CiA
G
CiA 500 500
500 500
0 0 0 0
0 20 40 60 80 0 20 40 60 80
Minutes
FIG 5 Reductase activity of HcrB on several hydroxycinnamic acids. HPLC

chromatograms of E. coli BL21(DE3) (pURI3-Cter) (control) or E. coli BL21(DE3)
(pURI3-Cter-hcrB) (HcrB) cell free extracts incubated at 37 ºC during 16 h. The
hydroxycinnamic acids assayed were p-coumaric (pCA), m-coumaric (mCA), o-
coumaric (oCA), ferulic acid (FA), caffeic acid (CA), and sinapic acid (SA). A non-
hydroxy derived cinnamic acid was used as control (cinnamic acid, CiA). The
corresponding reduced acids, such as phloretic acid (PhlA), 3-(3-hydroxyphenyl)
propionic (3-HPPA), 3-(2-hydroxyphenyl) propionic (2-HPPA) or melilotic,
hydroferulic (HFA), and hydrocaffeic (HCA), or hydrosinapic acid (HAS) detected are
indicated. Chromatograms were recorded at 280 nm.
Figure 6

16
*
Relative transcription level
14
12
*
10
*
8 *
*
6 * *
* ** *
* ** ** *
4 * *
*
* * **
2
0
p-Coumaric m-Coumaric o-Coumaric Ferulic Caffeic Sinapic Cinnamic Gallic
acid acid acid acid acid acid acid acid
FIG 6 Relative transcriptional expression of hcr genes in L. plantarum WCFS1 in

response to the presence of several hydroxycinnamic acids. L. plantarum cultures were
exposed during 10 min at 1.5 mM of a hydroxycinnamic acid (p-coumaric, m-coumaric,
o-coumaric, ferulic, caffeic, or sinapic acid). As controls, a non-hydroxy derived
cinnamic acid (cinnamic acid) and a hydroxybenzoic acid (gallic acid) were assayed.
Expression levels were calculated with the 7500 Fast System relative quantification
software using L. plantarum ldh gene as endogenous gene and the growth in the absence
of acid as growth condition calibrator. Expression level of hcrR, hcrA, hcrB and hcrC
genes are represented by brick-like shading, black colour, grey colour, and dotted-like
shading bars, respectively. The experiments were done in triplicate. The mean value and
the standard error are shown. Asteriks indicate a p value <0.1.
Figure 7
A kDa
1 2 3 4
87 kDa
66-
45-

31-
22 kDa
B 1 2 3 4 5 6
kDa
66- 87 kDa
57 kDa
45-
31-
22 kDa
21-
FIG 5 SDS-PAGE analysis of the production and purification of HcrA, HcrB, and
HcrAB recombinant proteins. (A) Soluble cell extracts of IPTG-induced E. coli
BL21(DE3) (pURI3-Cter) (lane 1), E. coli BL21(DE3) (pURI3-Cter-HcrA) (lane 2), E.
coli BL21(DE3) (pURI3-Cter-HcrB) (lane 3), and E. coli BL21(DE3) (pURI3-Cter-
HcrAB) (lane 4). (B) Purification of HrcA (lane 2), HcrB (lane 4) and HcrAB (lane 6)
recombinant proteins from the corresponding E. coli BL21cell extracts [pURI3-Cter-
HcrA (lane 1), pURI3-Cter-HcrB (lane 3), and pURI3-Cter-HcrAB (lane 5)]. HcrA
protein (22 kDa), HcrB (87 kDa), and hydrolyzed-HcrB (57 kDa).
ATCC BAA-350 (NZ_ASWH00000000.1), Streptococcus parasanguinis ATCC 15912
(NC_015678.1), Streptococcus uberis ATCC BAA-854 (NC_012004.1), Streptococcus
equinus ATCC 9812 (NZ_JNLO00000000.1), Streptococcus gallolyticus ATCC 43143
(NZ_CP018822.1), Streptococcus mutans ATCC 700610 (NC_004350.2), Clostridium
beijerinckii ATCC 51743 (NZ_CP010086.2), and Clostridium saccharobutylicum DSM
13864 (NC_022571.1). Arrows indicate genes. Genes having putative identical

functions are represented by identical shading. The genes having brick-like shading
encode putative transcriptional regulators. Genes coding for putative reductases similar
to HcrA and HcrB are represented by black and grey arrows, respectively.
Figure 8
Lactobacillus plantarum WCFS1
Lactobacillus paraplantarum DSM 10667
Lactobacillus pentosus DSM 20314
Lactobacillus fabifermentans DSM 21115

Lactobacillus alimentarius DSM 20249
Lactobacillus paralimentarius DSM 13961
Lactobacillus dextrinicus DSM 20335
Enterococcus phoeniculicola ATCC BAA-412
Enterococcus columbae DSM 7374
Enterococcus flavescens ATCC 49996
Enterococcus casseliflavus ATCC 12755
Enterococcus gilvus ATCC BAA-350
Clostridium beijerinckii ATCC 51743
Clostridium saccharobutylicum DSM 13864
Streptococcus parasanginis ATCC 15912
Streptococcus uberis ATCC BAA-854
Streptocoocus equinus ATCC 9812
Streptococcus gallolyticus ATCC 43143
Streptococcus mutans ATCC 700610
FIG 8 Genetic organization of hydroxycinnamate reductases from several lactic acid

bacteria, such as Lactobacillus plantarum WCFS1 (NC_004567.2), Lactobacillus
paraplantarum DSM 10667 (NZ_CP013130.1), Lactobacillus pentosus DSM 20314
(GCA_001433755.1), Lactobacillus fabifermentans DSM 21115
(NZ_AYGX00000000.2), Lactobacillus alimentarius DSM 20249
(NZ_AZDQ00000000.1), Lactobacillus paralimentarius DSM 13961
(NZ_AZDH00000000.1), Lactobacillus dextrinicus DSM 20335
(NZ_AYYK00000000.1), Enterococcus phoeniculicola ATCC BAA-412
(NZ_ASWE00000000.1), Enterococcus columbae DSM 7374
(NZ_AHYW00000000.1), Enterococcus flavescens ATCC 49996 (NC_020995.1),
Enterococcus casseliflavus ATCC 12755 (GCA_000191365.1), Enterococcus gilvus
Figure 9
A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
kb
7.7-
4.2-
2.7-

1.5-
0.9-
B
2000 2000 2000 2000
1 2
400 400
control
1000 1000 3-HPPA mCA
mCA 200 200
1000 1000
0 0
0 2000 0
20004000 0
400
3 4 5
0 20 40 60 80
500 500
mCA 1000 mCA 1000200 3-HPPA 200
250 250
0
0 2000 0 20000
2000 0
2000
20000
7 8
2000 0 20 40 60 80
6
0 20 40 60 80
mCA 1000 mCA 1000

1000 mCA 1000
1000 1000
mAU
0 0 0 0
0 200400
0 400
11
2000
0 20 40
mCA
60
9
80
10 300 300
3-HPPA 200 3-HPPA 200

1000 100200 200
100 100
0 0
4000 0 4000 0
400
14
400
12 13 300 300
3-HPPA 3-HPPA 200 200

3-HPPA 200
200 200200
100 100
0
2000 2000 0
0 0 0 0
15 16 17
0 20 40 60 80 0 20 40 60 80 400 20 40 60 80 400
1500 1500
1000 mCA 1000

1000 mCA 1000 3-HPPA mCA
200 200
500 500
0 0 0 0 0 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
Minutes Time (min)
FIG 9 Hydroxycinammate reductase activity in lactic acid bacteria. (A) PCR

amplification of the HcrB gene. Chromosomal DNA from several lactic acid bacteria
was used for PCR amplification with oligonucleotides 1655 and 1656 to amplify 1.4 kb
of the hcrB gene. PCR products were subjected to gel electrophoresis and stained with
Gel Red. Left lane, λ/EcoT14I (Takara) molecular size marker. Numbers indicate some
of the molecular sizes. (B) HPLC chromatograms of supernatants from lactic acid
bacteria grown during 10 days at 30 ºC in MRS media supplemented with 1.5 mM m-
coumaric acid. The m-coumaric acid (mCA) and 3-(3-hydroxyphenyl) propionic acid (3-
HPPA) detected are indicated. Chromatograms were recorded at 280 nm. The strains
assayed were Enterococcus casseliflavus DSM 20680 (1), E. durans DSM 20633 (2), E.
faecalis DSM 20478 (3), E. faecium CECT 4102 (4), E. gallinarum DSM 24841 (5), E.

hirae DSM 20160 (6), Lactobacillus brevis CECT 5354 (7), L. fermentum CECT 4007
(8), L. fructivorans CECT 4785 (9), L. paraplantarum DSM 10641 (10), L. plantarum
subsp. argentoratensis DSM 16365 (11), L. plantarum subsp. plantarum ATCC 14917
(CECT 748) (12), L. plantarum DSM 10492 (13), L. pentosus DSM 16366 (14), L.
sakei subsp. carnosus DSM 15831 (15), Leuconostoc citreum CECT 4025 (16), y
Streptococcus gallolyticus UCN34 (17).
Figure 10
CH 2

B
HO O
R2
PDC R1
A (Lp_3665)
R2 HO O
R1 C
HcrAB
(Lp_1424 / Lp_1425)
R2
R1
FIG 10 Schematic representation of hydroxycinnamic acid metabolism in L. plantarum

WCFS1. When R1 (OH) and R2 (H), the represented compounds are p-coumaric acid
(A), vinyl phenol (B), and phloretic acid (C). When R1 (OH) and R2 (OH), caffeic acid
(A), vinyl catechol (B), and hydrocaffeic acid (C). When R1 (OH) and R2 (OCH3), the
compounds are ferulic acid (A), vinyl guaiacol (B), and hydroferulic acid (C). When R1
(H) and R2 (OH), only the reduction reaction is carried out and the represented
compounds are m-coumaric acid (A), and 3-(3-hydroxyphenyl) propionic acid (C).

Amelia PDF

Hochgeladen von

Dokumentinformationen

Originaltitel

Copyright

Verfügbare Formate

Dieses Dokument teilen

Dokument teilen oder einbetten

Freigabeoptionen

Stufen Sie dieses Dokument als nützlich ein?

Sind diese Inhalte unangemessen?

Copyright:

Verfügbare Formate

Amelia PDF

Hochgeladen von

Copyright:

Verfügbare Formate

AEM Accepted Manuscript Posted Online 18 May 2018

Appl. Environ. Microbiol. doi:10.1128/AEM.01123-18

2 Unravelling the reduction pathway as alternative metabolic

3 route to hydroxycinnamate decarboxylation in Lactobacillus

Downloaded from http://aem.asm.org/ on September 22, 2019 by guest

12 Laboratorio de Biotecnología Bacteriana, Instituto de Ciencia y Tecnología de Alimentos y

13 Nutrición, ICTAN-CSIC, Madrid, Spain

17 Address correspondence to Rosario Muñoz, rmunoz@ictan.csic.es

22 LACTOBACILLUS PLANTARUM HYDROXYCINNAMATE REDUCTASE

25 plant-food fermentations where hydroxycinnamic acids are abundant. L. plantarum

26 efficiently decarboxylates these compounds, and also reduces them, yielding

Downloaded from http://aem.asm.org/ on September 22, 2019 by guest

28 induced by a hydroxycinnamic acid, the enzymatic machinery responsible for this

30 the transcriptomic response of L. plantarum to p-coumaric acid revealed a marked

32 In this work, disruption of these genes abolished the hydroxycinnamate reductase

33 activity of L. plantarum, supporting their involvement in such chemical activity.

34 Functional in vitro studies reveal that Lp_1425 (HcrB) exhibits hydroxycinnamate

37 formation of an active heterodimer (HcrAB) was observed. Since L. plantarum

38 reductase activity was only observed on hydroxycinnamic acids (o-coumaric, m-

43 that L. plantarum hydroxycinnamate reductase is a heterodimeric NADH-dependent

44 coumarate reductase acting on a carbon-carbon double bond.

48 Lactobacillus plantarum is a bacterial species frequently found in the fermentation of

49 vegetables where hydroxycinnamic acids are present. The bacterial metabolism on

Downloaded from http://aem.asm.org/ on September 22, 2019 by guest

51 activity of hydroxycinnamates. L. plantarum strains exhibit an as yet unknown

52 reducing activity, transforming hydroxycinnamates to substituted phenylpropionic

54 machinery involved in hydroxycinnamate reduction, HcrAB, was genetically

55 identified and characterized. The heterodimeric NADH-dependent coumarate

57 metabolic response to counteract the stressful conditions generated by food phenolics.

Downloaded from http://aem.asm.org/ on September 22, 2019 by guest

65 hydroxycinnamic acids could be transformed during the fermentation of plant-based foods.

71 abundant (2). Previous studies on L. plantarum demonstrated the existence of two

72 metabolic pathways for the transformation of hydroxycinnamates. The most efficient

77 derivatives (ethylphenol, ethylcatechol, and ethylguaiacol).

78 The second metabolic pathway for the transformation of hydroxycinnamates in L.

80 ferulic acids) to their corresponding phenylpropionic substituted acids [phloretic, 3-(3-

81 hydroxyphenyl) propionic acid (3-HPPA), dihydrocaffeic, and dihydroferulic acids] (6).

Downloaded from http://aem.asm.org/ on September 22, 2019 by guest

94 pathway remains biochemically and genetically uncharacterized.

95 Presently, many oxidoreductases of the class EC 1.3.1 acting on carbon-carbon

98 reductase subgroup (EC 1.3.1.11) only includes a NADH-dependent reductase partially

Downloaded from http://aem.asm.org/ on September 22, 2019 by guest

117 oxidorreductase type acting on the hydroxycinnamate carbon-carbon double bond in L.

118 plantarum remain largely unknown.

124 RESULTS AND DISCUSSION

126 Identification of the enzyme responsible for hydroxycinnamate reduction in L.

130 lp_1425, which would encode a fumarate reductase/succinate dehydrogenase, FAD-binding

Downloaded from http://aem.asm.org/ on September 22, 2019 by guest

136 knockout mutants were constructed by an insertion-duplication strategy. The correct

150 of hydroxycinnamate reduction, their genetic organization and expression profile in L.

Downloaded from http://aem.asm.org/ on September 22, 2019 by guest

160 HcrR, a LysR-type transcriptional regulator, in the reduction process.

166 (Fig. 3).

168 protein is a minimum requirement for hydroxycinnamate reductase activity in L. plantarum

176 recombinant plasmids pURI3-Cter-HcrA, pURI3-Cter-HcrB, and pURI3-Cter-HcrAB were

177 verified by DNA sequencing.

Downloaded from http://aem.asm.org/ on September 22, 2019 by guest

192 mM) no reduction of m-coumaric acid was detected (Fig S2B).

201 reaction (Fig. 4B).

Downloaded from http://aem.asm.org/ on September 22, 2019 by guest

216 coumaric acid to 3-HPPA, o-coumaric acid to melilotic acid or 3-(2-hydroxyphenyl)

222 coumaric, m-coumaric, p-coumaric, caffeic, ferulic, and sinapic acids).

Downloaded from http://aem.asm.org/ on September 22, 2019 by guest