Patent Application
20240132921
This application claims the benefit of European Patent Applications EP21155780.6, filed 8 Feb. 2021, and EP21196276.6 filed 13 Sep. 2021 and of the Portuguese Patent Application 20211000027222, filed 13 Jul. 2021, all of which are incorporated herein by reference.
The invention relates to a method for production of tyrosol, wherein a transgenic bacterial cell that heterologously expresses phenylpyruvate decarboxylase and that overexpresses phospho-2-dehydro-3-deoxyheptonate and prephenate dehydrogenase, and wherein pheAL and feaB are both inactivated or removed, is grown in a medium comprising a metabolic precursor of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P), particularly glucose, and optionally, phenylalanine as a supplement; and tyrosol is extracted from said medium. The invention also relates to a method for production of salidroside, wherein the transgenic cell additionally heterologously expresses uridine diphosphate dependent glycosyltransferase (UGT85A1, EC:2.4.1.).
Tyrosol is a phenolic compound of great industrial value and is marketed as a fine chemical.
Salidroside is a glucoside of tyrosol and has been studied as one of the potential compounds responsible for its putative antidepressant and anxiolytic actions.
Tyrosol concentration in plants is usually low, which leads to low commercial product yields and high production costs. Moreover, the natural extraction process for obtaining high purity tyrosol from plants is complex, which also makes the yield relatively low. Despite its natural abundance, because the cost of its extraction from natural sources is very high, tyrosol is also produced via chemical synthesis methods for industrial purposes, but these methods leave much room for improvement from a commercial point of view.
Transgenic cell as referred to in the current context means that the cell comprises at least one gene derived from a different organism than the host cell (referred to in the current specification as the transgene). This gene is introduced into the transgenic host cell via molecular biology methods.
Heterologous expression or heterologously expresses in relation to a certain gene as referred to in the current specification means that the gene is derived from a source other than the host species in which it is said to be heterologously expressed.
Overexpressing or overexpression in relation to a certain gene as referred to in the current specification means: addition of a functional (transgene or autologous) version of said gene, and/or addition of a promoter sequence controlling the autologous (native) version of said gene, leading to a significantly higher expression of the gene's biological activity relative to the wild-type (bacterial) cell. Significantly higher expression of the gene's biological activity means that there are at least 1.5-fold, particularly at least two-fold, the number of mRNA molecules inside the bacterial cell, compared to the wild-type bacterial cell. The overexpressed gene may also comprise mutations (substitutions, deletions and/or insertions) compared to the wild type nucleic acid and amino acid sequence. The mutations may increase the enzymatic efficacy, optimize the expression rate or change the enzymatic specificity.
Inactivation or knock-out in relation to a certain gene as referred to in the current specification means that the expression of that gene is significantly reduced, particularly by at least 30-fold, more particularly by at least 100-fold, compared to the wild-type bacterial cell or there is no gene expression of that gene.
Recombinant gene expression in relation to a certain gene as referred to in the current specification means: The recombinant gene is inserted into the host cell by molecular biology methods. The recombinant gene may originate from the same organism as the host cell, or from a different organism.
Supplement refers to amounts of a compound which are not the main carbon source for the bacterial cell, but are given in sufficient amounts that the cell's metabolism can compensate for auxotrophy of the compound. Phenylalanine is needed to cover the auxotrophy of pheAL deletion strains. The inventors used M9Y as it has yeast extract as a source of phenylalanine. Supplementation is needed either with yeast extract or pure phenylalanine.
A first aspect of the invention relates to a method for production of tyrosol, wherein a transgenic bacterial cell that heterologously expresses the following enzyme:
In certain embodiments, the transgenic bacterial cell is of the genus Escherichia, In certain embodiments, the transgenic bacterial cell is of the species E. coli. In certain embodiments, the transgenic bacterial cell is of the strain E. coli BL21.
In certain embodiments, the gene encoding the phenylpyruvate decarboxylase originates from yeast. In certain embodiments, the gene encoding the phenylpyruvate decarboxylase originates from S. cerevisiae.
A second aspect of the invention relates to a method for production of salidroside, wherein
A third aspect of the invention relates to a method for production of hydroxytyrosol, wherein a transgenic bacterial cell that heterologously expresses the following enzyme:
An alternative of the third aspect of the invention relates to a method for production of hydroxytyrosol, wherein a transgenic bacterial cell that recombinantly expresses each of the following enzymes:
In certain embodiments, the gene encoding the 4-hydroxyphenylacetate 3-monooxygenase originates from Escherichia. In certain embodiments, the gene encoding the 4-hydroxyphenylacetate 3-monooxygenase originates from E. coli.
In certain embodiments, the gene encoding the 4-hydroxyphenylacetate 3-monooxygenase comprises amino acid substitutions S210T, A211L and Q212E.
In certain embodiments of the third aspect, the medium comprises 5-10 g/L Na2HPO4·2H2O, 2-4 g/L KH2PO4, 0.25-1 g/L NaCl, 0.5-1.5 g/L NH4Cl, 1-3% (w/v) glucose, 0.01-0.05% (w/v) yeast extract, 3-7 mM MgSat, 0.005-0.02 g/L CaCl2), 0.5-2.0 g/L ascorbic acid, and antibiotics.
In certain embodiments of the third aspect, dodecanol is added to the medium. In certain embodiments of the third aspect, ˜25% dodecanol (v/v) is added to the medium. As dodecanol is immiscible with water it builds a second layer on top of the culture medium.
In certain embodiments of the third aspect, the cells are grown with >2% (v/v) of 02. In certain embodiments of the third aspect, the cells are grown with 2-4% (v/v) of 02.
In certain embodiments, the gene encoding uridine diphosphate dependent glycosyltransferase originates from a plant. In certain embodiments, the gene encoding uridine diphosphate dependent glycosyltransferase originates from Arabidopsis. In certain embodiments, the gene encoding uridine diphosphate dependent glycosyltransferase originates from A. thaliana.
In certain embodiments, the transgenic bacterial cell does not overexpress any of the following proteins:
In certain embodiments, the only transgenes of the transgenic bacterial cell are the ones mentioned above.
In certain embodiments, the overexpressed genes and the transgenes are introduced into the transgenic bacterial cell via one or several plasmid vector(s), particularly wherein
In certain embodiments, said transgenic bacterial cell comprises one or more plasmids encoding said heterologously expressed or overexpressed enzymes under control of a promoter sequence operable in said cell, particularly a T7 promoter (SEQ ID NO. 31), a lac promoter (SEQ ID NO. 32), a tac promoter (SEQ ID NO. 33) or a trc promoter (SEQ ID NO. 34), more particularly wherein
In certain embodiments, the expression of said heterologous and/or overexpressed genes is induced by adding isopropyl-β-d-thiogalactopyranoside (IPTG), particularly at a concentration of ˜0.1 mM IPTG for 96 h.
In certain embodiments, said medium comprises 10 to 50 g/L of glucose, particularly 15 to 30 g/L of glucose.
In certain embodiments, the transgenes are codon-optimized for expression in said transgenic bacterial cell.
In certain embodiments, the medium comprises 5-10 g/L Na2HPO4·2H2O, 2-4 g/L KH2PO4, 0.25-1 g/L NaCl, 0.5-1.5 g/L NH4Cl, 1-3% (w/v) glucose, 0.01-0.05% (w/v) yeast extract, 3-7 mM MgSO4, 0.005-0.02 g/L CaCl2) and antibiotics, particularly the antibiotics are 50-200 μg/mL ampicillin, 10-50 μg/mL kanamycin and 25-45 μg/mL chloramphenicol.
In certain embodiments, the cell is grown at 22° C. to 30° C., particularly at ˜30° C.
In certain embodiments, the protein phenylpyruvate decarboxylase has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or >95% sequence identity with SEQ ID NO 1 and has a catalytic activity of at least 75% of the activity of SEQ ID NO 1. In certain embodiments, the protein phospho-2-dehydro-3-deoxyheptonate aldolase has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or >95% sequence identity with SEQ ID NO 2 and has a catalytic activity of at least 75% of the activity of SEQ ID NO 2. In certain embodiments, the protein prephenate dehydrogenase has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or >95% sequence identity with SEQ ID NO 3 and has a catalytic activity of at least 75% of the activity of SEQ ID NO 3. In certain embodiments, the protein uridine diphosphate dependent glycosyltransferase has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or >95% sequence identity with SEQ ID NO 4 and has a catalytic activity of at least 75% of the activity of SEQ ID NO 4. In certain embodiments, the protein 4-hydroxyphenylacetate 3-monooxygenase has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or >95% sequence identity with SEQ ID NO 035 and has a catalytic activity of at least 75% of the activity of SEQ ID NO 035.
A fourth aspect of the invention relates to a transgenic cell as specified in any one of the above stated embodiments.
An alternative of the fourth aspect relates to a transgenic cell that heterologously expresses the following enzyme:
Another alternative of the fourth aspect relates to a transgenic cell that heterologously expresses each of the following enzymes:
Another alternative of the fourth aspect relates to a transgenic cell that heterologously expresses the following enzyme:
In certain embodiments of the fourth aspect, the transgenic bacterial cell is of the genus Escherichia, particularly wherein the transgenic bacterial cell is of the species E. coli, more particularly wherein the transgenic bacterial cell is of the strain E. coli BL21.
In certain embodiments of the fourth aspect, the gene encoding the phenylpyruvate decarboxylase originates from yeast, particularly from S. cerevisiae.
In certain embodiments of the fourth aspect, the gene encoding the 4-hydroxyphenylacetate 3-monooxygenase originates from Escherichia, particularly from E. coli.
In certain embodiments of the fourth aspect, the gene encoding the 4-hydroxyphenylacetate 3-monooxygenase comprises amino acid substitutions S210T, A211L and Q212E.
In certain embodiments of the fourth aspect, the gene encoding uridine diphosphate dependent glycosyltransferase originates from a plant, particularly from Arabidopsis, more particularly from A. thaliana.
In certain embodiments of the fourth aspect, the transgenic bacterial cell does not overexpress any of the following proteins:
In certain embodiments of the fourth aspect, the only transgenes of the transgenic bacterial cell are the ones mentioned above.
In certain embodiments of the fourth aspect, the overexpressed genes and the transgenes are introduced into the transgenic bacterial cell via one or several plasmid vector(s), particularly wherein
In certain embodiments of the fourth aspect, said transgenic bacterial cell comprises one or more plasmids encoding said heterologously expressed or overexpressed enzymes under control of a promoter sequence operable in said cell, particularly a T7 promoter (SEQ ID NO. 31), a lac promoter (SEQ ID NO. 32), a tac promoter (SEQ ID NO. 33) or a trc promoter (SEQ ID NO. 34), more particularly wherein
In certain embodiments of the fourth aspect, the protein phenylpyruvate decarboxylase has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or >95% sequence identity with SEQ ID NO 1 and has a catalytic activity of at least 75% of the activity of SEQ ID NO 1. In certain embodiments of the fourth aspect, the protein phospho-2-dehydro-3-deoxyheptonate aldolase has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or >95% sequence identity with SEQ ID NO 2 and has a catalytic activity of at least 75% of the activity of SEQ ID NO 2. In certain embodiments of the fourth aspect, the protein prephenate dehydrogenase has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or >95% sequence identity with SEQ ID NO 3 and has a catalytic activity of at least 75% of the activity of SEQ ID NO 3. In certain embodiments of the fourth aspect, the protein uridine diphosphate dependent glycosyltransferase has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or >95% sequence identity with SEQ ID NO 4 and has a catalytic activity of at least 75% of the activity of SEQ ID NO 4. In certain embodiments of the fourth aspect, the protein 4-hydroxyphenylacetate 3-monooxygenase has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or >95% sequence identity with SEQ ID NO 035 and has a catalytic activity of at least 75% of the activity of SEQ ID NO 035.
The present specification also comprises the following items.
Items
Cloning Strategy
E. coli DH5a cells (New England BioLabs, Massachusetts, USA) were used for gene cloning and vector propagation. This strain was cultured in Luria-Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) with the appropriate antibiotic concentration. The solid version of this medium included 20 g/L of agar. All cultivations were performed at 37° C. and, in the case of liquid cultures, under shaking conditions (200 rpm). For long-term storage, glycerol was added to a final concentration of 30% (v/v) to overnight cultures in selective media and kept in a −80° C. freezer.
The genes used in this study were amplified by polymerase chain reaction (PCR) using Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Waltham, USA) in a LifeECO Thermal Cycler. All primers were purchased from Integrated DNA Technologies (Coralville, USA). DNA fragments were purified using DNA Clean and Concentrator DNA Kit (Zymo Research, Irvine, USA).
Plasmids were extracted using Plasmid Miniprep Kit (Zymo Research). All digestions were performed using the appropriate FastDigest® restriction endonucleases (Thermo Scientific). Ligations were performed with T4 DNA Ligase (Thermo Scientific) and transformed in chemically competent E. coli DH5a cells and E. coli BL21 (DE3) using Mix & Go E. coli Transformation Kit & Buffer Set (Zymo Research). The success of ligation was checked through colony PCR using DreamTaq (Thermo Scientific) and further confirmed by sequencing (StabVida, Lisbon, Portugal). Protocols were performed in accordance with manufacturer's instructions.
The tyrAfbr gene and the codon-optimized genes ScARO10*, KpPDC, EipdC and AtUGT85A1 were purchased from IDT DNA Technology (Coralville, USA) and cloned in pET-21a(+) vector (Novagen, Darmstadt, Germany) in the case of tyrAfbr and ScARO10*, in pJET1.2 vector (CloneJET PCR Cloning Kit, Thermo Scientific) in the case of KpPDC and EipdC, and in pET-28a(+) vector (Novagen, Darmstadt, Germany) for the case of UGT gene. aroFfbr and hpaBC* genes were amplified from E. coli BL21 (DE3) genomic DNA from New England BioLabs (Massachusetts, USA). hpaBC* gene was mutated in S210T, A2111L and Q212E of HpaB subunit, in order to improve the activity for tyrosol (Chen, 2019). adhP* was kindly provided by Prof. Isabel Rocha group (University of Minho, Portugal).
Plasmid Construction and Bacterial Strains
The plasmids pET-21a(+), pET-28a(+), pACYCDuet and pRSFDuet (Novagen, Darmstadt, Germany) were used to provide individual expression of each protein under the control of the T7lac promoter and a ribosome binding site (RBS). All the plasmids were constructed by traditional molecular biology techniques and the success of the plasmid constructions was confirmed by colony PCR and sequencing the regions of interest with the appropriate primers.
E. coli DH5a was used as a host for gene cloning and plasmid propagation while E. coli BL21 (DE3), the parent strain, was engineered to produce tyrosol, salidroside, and hydroxytyrosol. For all the strains, positive transformants were isolated in LB agar plates, containing the appropriate antibiotic concentrations (100 μg/mL ampicillin, 30 μg/mL kanamycin and 34 μg/mL chloramphenicol) and incubated at 37° C., overnight. To confirm the success of the transformation, a few transformant colonies were cultivated in LB medium with appropriate antibiotics, overnight. Afterwards, plasmids were extracted, digested with appropriate restriction enzymes and the correct fragment lengths were confirmed by running the digestion in a 1% (w/v) agarose gel.
Construction of Tyrosol Plasmids and Strains
The plasmid pET-21a(+) (Novagen), with ampicillin resistance marker, was used to clone the genes adhP*, aroFfbr, tyrAfbr and the codon-optimized gene, ScARO10*. The optimized phenylpyruvate decarboxylase gene ScARO10* was amplified by PCR using the primer pair ARO10*_pet_fw/ARO10*_RBS_rev (primers are shown in Table 1) and the plasmid pET-21a(+) was amplified by PCR using the primer pair pet21a_fw/pet21a_rev. These two fragments were fused using circular polymerase extension cloning (CPEC) (Quan, J. et al, Nat Protoc 6, 242-251 (2011)). Then, this PCR product was amplified by PCR using the primers ARO10*_pet_fw and ARO10_hindiii_rev, restricted with Ndel and Hindlll and cloned into the plasmid pET-21a(+), also restricted with these enzymes, originating pET-21a(+)_ScARO10*. The PCR product for aroFfbr, with the mutation D147N, was amplified by PCR in two fragments, using the primer pairs aroF_fbr_RBS_fwlaroF_D147N_rev and aroF_D147N_fwlaroF_fbr_RBS_rev. These two fragments were fused using PCR technique with the primer pair aroF_fbr_RBS_fwlaroF_fbr_RBS_rev, and was restricted and ligated into Hindlll and Notl restriction sites of the previous construction, originating pET-21a(+)_ScARO10*_aroFfbr. The chorismate mutase or prephenate dehydrogenase gene, tyrAfbr, with the mutations M531 and A354V was ordered from IDT DNA Technology (USA) and restricted with Notl and Xhol in order to be cloned into the previous construction, originating pET-21a(+)_ScARO10*_aroFfbr_tyrAfbr. The alcohol dehydrogenase gene, adhP*, was amplified by PCR from the plasmid pET-28a(+)_adhP*, that was kindly provided by Prof. Isabel Rocha group (University of Minho, Portugal) with the primers Tyr2_adhp_JO_fw and Tyr2_adhp_JO_rev, after the plasmid pET-21a(+)_ScARO10*_aroFfbr_tyrAfbr was restricted with Notl and then the amplified fragment and the plasmid were ligated using the In-Fusion® HD Cloning Plus Kit (TaKaRa, France), forming pET-21a (+)_ScARO10* aroFfbr_adhP*_tyrAfbr.
ggtaccTAATAGAAATAATTTTGTTTAACTTTAtaaggaggaaaaaaa
Alternatively, the plasmid pET-28a(+) (Novagen), containing kanamycin resistance gene, was also used to clone the genes aroFfbr and tyrAfbr. For that, the pET-28a(+) plasmid was amplified by PCR using the primers pet21a_fw and pet28a_RBS_rev and the aroFfbr gene was amplified from pET-21a(+)_ScARO10*_aroFfbr_tyrAfbr plasmid, using the primers RBS_linker_st7_fw and aroF_fbr_RBS_rev. After, both fragments were merged using CPEC, originating pET-28a(+)_aroFfbr. Afterwards, this plasmid was amplified by PCR with the primers pet21a_fw and aroF_fbr_RBS_rev and the tyrAfbr gene was amplified from pET-21a(+)_ScARO10*_aroFfbr_tyrAfbr plasmid, using the primers RBS_linker_st7 fw and tyrA_fbr pet rev. Finally, these two fragments were fused using the CPEC strategy, forming pET-28a(+)_aroFfbr tyrAfbr.
Furthermore, two alternative decarboxylases encoded by EipdC and KpPDC genes from Enterobacter sp. and Komagataella phaffii, respectively, were tested instead of ScARO10*. For that, the synthetic genes previously cloned into pJET1.2 (Thermo Scientific) were restricted with Xbal and Hindlll and cloned into the plasmid pET-21a(+)_ScARO10*_aroFfbr_tyrAfbr, also restricted with these enzymes, originating pET-21a (+)_EipdC_aroFfbr tyrAfbr and pET-21a(+)_KpPDC_aroFfbr_tyrAfbr, respectively.
The plasmids and tyrosol production strains constructed and used in this work are listed in Table 2.
E. coli DH5α
E. coli BL21 (DE3)
E. coli BL21 (DE3) ΔpheALΔfeaB
E. coli BL21 (DE3) with knockouts in
E. coli BL21 (DE3) with
E. coli BL21 (DE3) with
E. coli BL21 (DE3) with
E. coli BL21 (DE3) with
E. coli BL21 (DE3) ΔpheALΔfeaB with
E. coli BL21 (DE3) ΔpheALΔfeaB with
Construction of Salidroside Plasmids and Strains
The plasmid pET-28a(+) was used to clone the codon optimized gene AtUGT85A1, corresponding to the final step of the proposed pathway, which consists in the conversion of tyrosol into salidroside. The AtUGT85A1 gene was amplified by PCR using the primers UGT85a1_ncoi_fw and UGT85A1_ (primers are shown in Table 3) with restriction sites to Ncol and BamHI and cloned in pET-28a(+), originating pET-28a(+)_AtUGT85A1.
Additionally, to test different plasmid copy number, the AtUGT85A1 gene was cloned in the plasmids pACYCDuet and pRSFDuet, with chloramphenicol and kanamycin resistance marker, respectively. To construct pACYCDuet_AtUGT85A1 and pRSFDuet_AtUGT85A1 plasmids the AtUGT85A1 gene was extract with Ndel and Xhol from pET28a(+)_AtUGT85A1 plasmid, and cloned in pACYCDuet and pRSFDuet, respectively, also digested with these enzymes.
Moreover, to increase salidroside production the T7lac promoter in pACYCDuet_AtUGT85A1 was replaced by trc promoter, using PCR technique with primers pacyc_trc_mc2_fw and pacyc_trc_mc2_rev, originating pACYCDuet_trc-promoter_AtUGT85A1.
The plasmids and salidroside production strains constructed and used in this study are listed in Table 4.
E. coli DH5α
E. coli BL21 (DE3)
E. coli BL21 (DE3) ΔpheALΔfeaB
E. coli BL21 (DE3) with knockouts in the
E. coli BL21 (DE3) with
E. coli BL21 (DE3) with
E. coli BL21 (DE3) with
E. coli BL21 (DE3) with pET-
E. coli BL21 (DE3) with
E. coli BL21 (DE3) with
E. coli BL21 (DE3) ΔpheALΔfeaB with
E. coli BL21 (DE3) ΔpheALΔfeaB with
Construction of Hydroxytyrosol Plasmids and Strains
The plasmid pET-28a(+) was used to clone the hpaBC* gene with mutations in S210T, A2111L and Q212E of HpaB subunit, which enzyme is responsible for conversion of tyrosol into hydroxytyrosol. These mutations, identified by Chen and his co-workers, improve the activity and specificity of HpaB towards tyrosol. The hpaBC* gene was amplified by PCR in two fragments to insert the given mutations using the primer pairs hpaB_rbs_xbailhpab_210_2_rev and hpab_210_2_fwlhpac_bamhi_rev, using genomic DNA of E. coli BL21 (DE3) as template (primers are shown in Table 5). These two fragments were fused using PCR technique with the primer pair hpaB_rbs_xbailhpac_bamhi_rev, restricted and ligated into Xbal and BamHI restriction sites of the plasmid pET-28a(+), forming pET-28a(+)_hpaBC*.
In addition, to test the influence of different plasmid copy number, the hpaBC*gene was cloned in the plasmids pACYCDuet and pRSFDuet with chloramphenicol and kanamycin resistance marker, respectively. For both cases, the hpaBC* gene was extract from pET-28a(+)_hpaBC* plasmid, restricted and ligated into Ndel and Xhol restriction sites of each plasmid, originating pACYCDuet_hpaBC* and pRSFDuet_hpaBC*.
The plasmids and hydroxytyrosol production strains constructed and used in this work are listed in Table 6.
E. coli DH5 α
E. coli BL21 (DE3)
E. coli BL21 (DE3) with
E. coli BL21 (DE3) with
E. coli BL21 (DE3) with
Strain Maintenance and Cultivation Media
All strains were cultivated in LB broth medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) and M9Y medium, which contained 1×M9 minimal salts (Na2HPO4·2H2O, 8.5 g/L; KH2PO4, 3.0 g/L; NaCl, 0.5 g/L; NH4Cl, 1.0 g/L) and 2% (w/v) glucose, and was supplemented with 0.025% (w/v) yeast extract, 5 mM MgSO4, 0.011 g/L CaCl2) and with the appropriate antibiotic concentrations (100 μg/mL ampicillin, 30 μg/mL kanamycin and 34 μg/mL chloramphenicol). Additionally, strains with background of E. coli BL21 (DE3) LpheALLfeaB were supplemented with 20 mg/L of phenylalanine.
A single colony of the engineered E. coli strain was used to inoculate 10 ml liquid LB medium containing appropriate antibiotics and allowed to grow overnight at 37° C. with agitation of 200 rpm. Then, the precultures were transferred to 250 mL shake flask with 50 mL of LB medium containing the appropriate antibiotic, with an initial optical density (00600) of 0.1. Firstly, the cultures were cultivated on a rotary shaker at 200 rpm and 37° C. until cell density (00600) reached 0.6-0.8. At this point, in the case of tyrosol and salidroside, cells were collected by centrifugation (6000 rpm for 10 min), resuspended in 50 ml M9Y medium with suitable antibiotics and the gene expression was induced with isopropyl 1-thio-β-D-galactopyranoside (IPTG) at a final concentration of 0.1 or 1 mM. After induction, the cultures were incubated at 22 or 30° C. and with agitation of 200 rpm. Samples of broth were collected at time 0, induction time 24, 48, 72, 96 and 121 h for HPLC analysis and cell density measurement. For hydroxytyrosol, cells were cultivated as stated above with some changes: a) addition of 1 g/L of ascorbic acid; b) addition or absence of 12.5 ml of 1-dodecanol to the growth medium at 16 h of induction. These formulations aimed to improve hydroxytyrosol recovery. Samples of broth were collected at time 0, induction time 24 and 48 for high-performance liquid chromatography (HPLC) analysis and cell density measurement. All the experiments were performed in triplicate and the samples were analysed by HPLC and nuclear magnetic resonance spectroscopy (NMR).
Analytical Methods
The tyrosol, salidroside, hydroxytyrosol, glucose and organic acids content of the fermentation medium were analysed using HPLC. NMR technique was used to confirm the presence of tyrosol, salidroside and hydroxytyrosol in the medium samples and for quantification of hydroxytyrosol in the 1-dodecanol fraction of the biphasic growth.
For each sampling, 1 mL of broth were removed from the culture and centrifuged at 15000 rpm for 10 min to separate cells from the medium. Next, the supernatant was filtered through a membrane filter with a pore size of 0.22 μm into HPLC vials and stored at −20° C. until further analysis. Tyrosol, salidroside and hydroxytyrosol concentrations were quantified by an HPLC apparatus from SHIMADZU (Kyoto, Japan) model Nexera X2 equipped with DAD SPD-M20A detector, also from SHIMADZU. The samples were analysed using a Kinetex® C18 column (150 mm×2.1 mm; particle size, 1.7 μm) from Phenomenex (California, USA). For the analysis of tyrosol and salidroside, a 5 μl sample of the fermentation supernatant was applied to the column, along with the mobile phases included solvent A (0.1% formic acid in H2O) and solvent B (acetonitrile with 0.1% formic acid). Each sample was eluted at 30° C., with a flow rate of 0.5 ml/min and under the following HPLC conditions: solvent B concentration was maintained at 5% for 1 min, then increased from 5% to 9% over 4 min, after increased from 9% to 30% during 5 min, remained at 30% for 6 min and finally decreased from 30% to 5% over 2 min. The compounds were detected at 280 nm. In these conditions, the retentions time of tyrosol and salidroside were 7 and 5 min, respectively. To quantify tyrosol and salidroside in the culture medium, calibration curves were generated with a series of known concentrations of the tyrosol standard (Fisher, USA) and salidroside standard (Sigma-Aldrich, USA) dissolved in water. The R2 coefficients for the calibration curves were >0.99. For the analysis of hydroxytyrosol, a 10 μl sample of the fermentation supernatant was applied to the column, along with the mobile phases included solvent A (0.5% acetic acid in H2O) and solvent B (100% acetonitrile). Each sample was eluted at 30° C., with a flow rate of 0.3 ml/min and under the following HPLC conditions: solvent B concentration was maintained at 5% for 2 min, then increased from 5% to 9% over 2 min, after increased from 9% to 30% over 6 min, then was maintained at 30% for 4 min and finally decreased from 30% to 5% over 2 min. The hydroxytyrosol was detected at 280 nm with the retention time of 8 min. To quantify hydroxytyrosol in the culture medium, calibration curves were generated with a series of known concentrations of the hydroxytyrosol standard (TCI, Japan) dissolved in water. The R2 coefficients for the calibration curves were >0.99.
Quantitative analysis of glucose and fermentation products were performed using HPLC apparatus from Jasco (Japan) model LC-Netll/ADC equipped with UV-2075 Plus and RI-4030 Plus detectors, also from Jasco. The samples were analysed using an Aminex HPX-87H column (300 mm×7.7 mm) from Bio-Rad (USA), which was kept at 60° C. and 0.5 mM H2504 was used as mobile phase with a flow rate of 0.5 mL/min. Glucose and ethanol were detected with a refractive index (RI) detector (4030, Jasco) and organic acids (acetate, formate, lactate, succinate and pyruvate) were detected at 210 nm using the UV detector. Calibration curves were obtained by injecting standards with known concentrations for each metabolite. Metabolite concentrations in samples were calculated by comparing the peak areas of the samples with the calibration curves. The R2 coefficients for the calibration curves were >0.99.
Hydroxytyrosol in the 1-dodecanol fraction of biphasic growth was quantified by a proton magnetic resonance spectroscopy (1H) using a NMR device apparatus from BRUKER (USA) model Avance II 400 MHz spectrometer. To do so, 300 μl of 1-dodecanol fraction was diluted in 300 μl of deuterated chloroform plus 5 μl of a 250 mM formate solution (internal standard). To confirm the production of tyrosol, hydroxytyrosol and salidroside, positive samples analysed in the HPLC were promptly transferred to an NMR tube with 10% (v/v) of D2O and read in the spectrometer referred above.
All cell optical density measurements (00600) were performed using the NanoDrop One spectrophotometer from Thermo Fisher (USA).
Statistical Analysis
All experiments were independently conducted three times. Experimental data are represented by the mean±standard deviation. Student's t test was used to conduct statistical analyses. Differences between engineered strains were considered significant when the P value was <0.05.
Sequences
Protein sequences:
Saccharomyces
cerevisiae
Escherichia coli
Escherichia coli
Arabidopsis
thaliana
Escherichia coli
Gene Sequences:
Escherichia
coli
Escherichia
coli
Arabidopsis
thaliana
Saccharomyces
cerevisiae
Escherichia
coli
Escherichia
coli
Escherichia
coli
Escherichia
coli
Promoter Sequence
The main goal of this study was the optimization of the bioprocess of production of tyrosol and its derivatives in E. coli to titers of gram per liter, since these compounds have high-added value and important biological activities and applications. To do so, E. coli BL21 (DE3) was engineered to produce tyrosol and salidroside through the pathway depicted in
The tyrosol biosynthesis pathway implemented in E. coli BL21 (DE3) (
The isopropyl-β-d-thiogalactopyranoside (IPTG) is an effective inducer of the powerful T7 and trc promoters and is commonly used in cloning procedures. To select the best IPTG concentration to induce tyrosol production strains, the strain ST93 was induced with 0.1 and 1 mM of IPTG in M9Y medium for 48 h. Under these conditions, the strain ST93 obtained 0.65±0.07 g/L and 0.21±0.01 g/L of tyrosol after induction with 0.1 and 1 mM of IPTG, respectively (Table 10). In that way, 0.1 mM of IPTG revealed to be the best concentration to induce tyrosol production strains.
Phenylpyruvate decarboxylase is an enzyme involved in the Ehrlich pathway and catalyses the decarboxylation of phenylpyruvate to phenylacetaldehyde (
The alcohol dehydrogenase AdhP*, that was kindly provided by Prof. Isabel Rocha group, can reduce 4-hydroxyphenylacetaldehyde into tyrosol and was modified to a better performance for large substrates (
Furthermore, to test the best conditions for AdhP* catalysis, the strain ST81 was induced with 0.1 mM of iPTG in M9Y medium at 22° C. for 48 h. Under these conditions, the strain ST81 could produce 0.29±0.02 g/L of tyrosol (
As stated before, endogenous AD H(s) in E. coli are capable of reducing 4-hydroxyphenylacetaldehyde into tyrosol, however this intermediary compound can also be oxidized into 4-hydroxyphenylacetate by an endogenous phenylacetaldehyde dehydrogenase, named FeaB (
Analysing these results, it is possible to verify that the addition of phenylalanine improves significantly the tyrosol production (p<0.001) on ST191 and decreases for ST170. Furthermore, growth of these strains behaves differently to the addition of phenylalanine, with improved parameters for ST170 and no response in the case of ST191, in comparison with growth with no phenylalanine. In conclusion, the best tyrosol titer from glucose achieved in this work is 1.41±0.02 g/L with strain ST191 corresponding to 10 mM and was attained after 96 h of induction with 0.1 mM of IPTG and addiction of 20 mg/L of phenylalanine in M9Y medium. This result corroborates the titer accomplished by Yang and his collaborators, whose strain produces 1.32 g/L of tyrosol from glucose after 48 h of induction with 0.6 mM of IPTG in M9Y medium by engineering E. coli MG1655 with heterologous expression of ScARO10* and knockout of feaB, pheA, tyrB and tyrR genes (Yang et al., Chinese Journal of Chemical Engineering, 26, 2615-2621). However, in this study the inventors produce 6% more tyrosol than Yang and his team with a strain harbouring ScARO10*, aroFfbr and tyrAfbr genes and with deletions of feaB and pheAL genes. Furthermore, the inventors verify that the heterologous expression of ScARO10* associated with the overexpression of aroFfbr and tyrAfbr in an operon-like system cloned in a pET system improves tyrosol production in approximately 92% in comparison with the first strain constructed (ST53). Additionally, the tyrosol production was enhanced in approximately 50% with the feaB and pheAL gene knockouts in comparison with the strain without these knockouts. On the other hand, AdhP* overexpression did not improve tyrosol production, on the contrary, it decreases 7% in comparison with the strain without this enzyme as discussed above.
Salidroside Production
Salidroside is a phenylethanoid glycoside that was widely distributed in the plant kingdom and has recently attracted increased attention because of its important role in the adaptogenic effect. During the last decade, new metabolic engineering approaches were implemented in E. coli, however more effective strategies are required.
The salidroside biosynthesis pathway created in E. coli BL21 (DE3) was achieved by heterologous expression of ScARO10* and AtUGT85A1 genes, and overexpression of aroFfbr and tyrAfbr genes in different plasmids. The critical step of this pathway is the glycosylation of tyrosol into salidroside mediated by uridine diphosphate dependent glycosyltransferase (UGT85A1). This gene was inserted into pET-28a(+) and transformed in E. coli BL21 (DE3) harbouring pET-21a(+)_ScARO10* and in E. coli BL21 (DE3) harbouring pET-21a(+)_ScARO10*_aroFfbr_tyrAfbr, achieving the strains ST95 and ST92, respectively. Both strains were grown aerobically in M9Y medium with glucose and showed a maximum of 0.02±0.01 g/L of salidroside and tyrosol after 48 h of induction with 1 mM of IPTG in M9Y medium for strain ST95 and overexpression of aroFfbr and tyrAfbr, while strain ST92 could produce ten-fold higher titer of salidroside than strain ST95, at the same conditions (0.24±0.05 g/L of salidroside and 0.13±0.03 g/L of tyrosol). This result supports the result obtained by strain ST93 for tyrosol production, which indicated that the overexpression of aroFfbr and tyrAfbr associated with the heterologous expression of ScARO10* enhanced tyrosol production and consequently, salidroside production by UGT85A1.
With the purpose of verifying if the induction with 0.1 mM of IPTG was also the best concentration for salidroside production, the strain ST92 was induced with 0.1 mM of IPTG for 48 h in M9Y medium. Under these conditions, the strain ST92 produces 0.41±0.07 g/L of salidroside and 0.15±0.04 g/L of tyrosol after 48 h of induction in M9Y medium (Table 11). This result demonstrated that, as well as for tyrosol production, salidroside production was significantly enhanced (p<0.001) by induction with 0.1 mM of IPTG instead of 1 mM of IPTG.
However, the strain ST92 metabolism exhibited a bottleneck in salidroside production as tyrosol is accumulated in both concentrations of IPTG that were tested. Different scenarios can explain this accumulation, such as: growth arrest by low pH, consequence of a fermentative metabolism lack of UDP-glucose or other critical nutrient depleted from the medium; or improper enzyme production/folding. Therefore, different M9Y medium compositions were tested in order to see the influence of glucose and pH in salidroside production. For that, the strain ST92 was induced with 0.1 mM of IPTG in M9Y with two-fold amount of salts (2×M9Y) and complemented with 5, 10 or 20 g/L of glucose for 48 h. Under these conditions, the strain ST92 could produce 0.10±0.00 g/L of salidroside and 0.08±0.00 g/L of tyrosol from 5 g/L of glucose, 0.26±0.00 g/L of salidroside and 0.12±0.02 g/L of tyrosol from 10 g/L of glucose, and 0.34±0.01 g/L of salidroside and 0.19±0.00 g/L of tyrosol from 20 g/L of glucose (Table 12). Regarding glucose supply, salidroside production was favoured by addiction of 20 g/L of glucose in 2×M9Y medium, although the best salidroside titer was achieved in M9Y medium complemented with 20 g/L of glucose (0.41±0.07 g/L. This result indicated that buffering the M9Y medium with addiction of two-fold amount of salts did not improve salidroside production.
On the other hand, the variation of medium pH was significantly higher in 2×M9Y medium complemented with 20 g/L of glucose (p<0.01) than in 2×M9Y medium supplemented with 5 and 10 g/L of glucose. This pH variation was caused by acetate production, which was higher when 2×M9Y medium was complemented with 20 g/L of glucose. Moreover, the pH variation in M9Y medium and 2×M9Y medium complemented with 20 g/L of glucose was not very significant (p<0.05). Taking all of these in consideration, the best conditions for salidroside production were induction with 0.1 mM of IPTG in M9Y medium complemented with 20 g/L of glucose.
Despite all the attempts for medium optimization, the bottleneck in salidroside production has not been overcome. Thereby, a new strategy was implemented in order to understand if changing the expression level of UGT85A1, by cloning it in different copy number plasmids, would have an effect in salidroside production (
Additionally, it was possible to verify that increasing the plasmid copy number (pACYCDuet<pET-28a(+)<pRSFDuet) the tyrosol conversion into salidroside was almost totally achieved, however the salidroside titer was not enhanced, indicating that possibly UGT85A1 would be insoluble. Taking this in consideration, the T7 promoter of pACYCDuet_AtUGT85A1 was replaced by trc promoter, originating the strain ST176, in order to optimize tyrosol conversion and salidroside titer. This strain could produce 1.64±0.07 g/L of salidroside and only 0.10±0.06 g/L of tyrosol after 48 h of induction with 0.1 mM of iPTG in M9Y medium (
To improve metabolic flow towards salidroside, the inventors set to clone the best two gene organizations and attempt to improve its production, the best gene organizations were cloned into E. coli BL21 (DE3) harbouring feaB and pheAL gene knockouts (
Once again, in ST178, tyrosol at a major extent is converted into salidroside and as observed before, ST172 accumulated salidroside in conjunction with significant amounts of tyrosol. In conclusion, cloning AtUGT85A1 in a low copy plasmid and under the influence of a weaker promoter balanced the production of the protein and improved significantly the salidroside titers. Furthermore, it was also possible to verify that the knockouts improved salidroside production in both strains, comparing to the respective strains without knockouts.
Besides that, the influence of phenylalanine supplementation was also evaluated on salidroside production. For that, the strains ST172 and ST178 were induced for 96 h with 0.1 mM of IPTG in M9Y medium supplemented with 20 mg/L of phenylalanine. Under these conditions, the strain ST172 could produce 0.43±0.01 g/L of salidroside and 0.90±0.03 g/L of tyrosol and the strain ST178 could produce 1.25±0.42 g/L of salidroside and 0.40±0.12 g/L of tyrosol (
Hydroxytyrosol Production
Hydroxytyrosol is one of the most abundant phenolic alcohols in olives and have some exceptional features that makes it ideal for implementation in the nutraceutical, agrochemical, cosmeceutical and food industry. However, besides all the work already done, a cost-effectively approach was not found yet.
The fundamental step in hydroxytyrosol biosynthesis is the conversion of tyrosol into hydroxytyrosol. To mediate this step there are several possible candidate enzymes described in literature. Espin and his team used a mushroom tyrosinase, however this enzyme is unstable and its activity is inhibited by phenols and ascorbic acid. Another study conducted by Liebgott and his co-workers demonstrated that 4-hydroxyphenylacetic acid 3-hydroxylase from different bacteria was responsible of converting tyrosol into hydroxytyrosol. Furthermore, other native hydrolases of some aromatic compound degrading microorganisms, such as Serratia marcescens, Pseudomonas aeruginosa, Pseudomonas putida F6 and Halomonas sp. strain HTB24 were identified to convert tyrosol into hydroxytyrosol. More recently, 4-hydroxyphenylacetate 3-monooxygenase (HpaBC*) was engineered from E. coli in order to improve its activity and specificity for tyrosol. With this engineered enzyme they achieved a high activity for tyrosol and founded that its docking energy for tyrosol was much lower than that for wild-type HpaBC. So, in this study, HpaBC* was selected from all enzymes since it is an endogenous enzyme of E. coli and was engineered for a better performance from tyrosol as a substrate. That way, the hydroxytyrosol biosynthesis pathway was implemented in E. coli BL21 (DE3) by heterologous expression of ScARO10* gene and overexpression of aroFfbr, tyrAfbr and hpaBC* genes (
As stated before, hydroxytyrosol is an antioxidant easily oxidized during its production, making this compound more unstable than tyrosol or salidroside. Besides that, it was reported that hydroxytyrosol shows an inhibitory effect on cell growth above 1 g/L. Taking this in consideration, the inventors designed a biphasic growth with 1-dodecanol that could sequester hydroxytyrosol, avoid its oxidation and cell toxicity. To do so, the inventors added 25% (v/v) of 1-dodecanol to the culture media when growth was no longer observed, which occurs 16 h after protein induction. Maximal production was detected at 48 h of induction with 0.1 mM of IPTG in M9Y medium supplemented with 1 g/L of ascorbic acid and addition of 12.5 ml of 1-dodecanol (
Such as for tyrosol and salidroside, different IPTG concentrations were tested to evaluate the best induction condition for hydroxytyrosol production. In this case, the strain ST119 were induced with 0.1 mM and 0.2 mM of IPTG for 48 h in M9Y medium supplemented with 1 g/L of ascorbic acid and addition of 12.5 ml of 1-dodecanol. The strain ST119 produced 0.56±0.09 g/L of hydroxytyrosol and trace amounts of tyrosol after induction with 0.2 mM of IPTG, which was significantly less than the hydroxytyrosol titer obtained when strain ST119 was induced with 0.1 mM of IPTG (0.92±0.15 g/L of hydroxytyrosol) (Table 13). Furthermore, the cell density (OD600 nm) was not affected when the cells were induced with 0.1 or 0.2 mM of IPTG despite the different accumulated amounts of hydroxytyrosol. With this result was possible to realise that the best conditions for hydroxytyrosol production were induction for 48 h with 0.1 mM of IPTG in M9Y medium supplemented with 1 g/L of ascorbic acid and addition of 12.5 ml of 1-dodecanol. To evaluate the solubility of ARO10*, AroFfbr, TyrAfbr and HpaBC* proteins whose genes were overexpressed in the pET system, a SDS-PAGE gel was performed which shows that overproduced proteins are mainly soluble.
In conclusion, the best condition for hydroxytyrosol production was 6 mM and was obtained with strain ST119, 48 h after induction with 0.1 mM of IPTG in M9Y medium supplemented with 1 g/L of ascorbic acid and 20 g/L of glucose and addition of 12.5 ml of 1-dodecanol. Under these conditions, it was possible to accumulate 0.92±0.15 g/L of hydroxytyrosol, which corresponds to an increase of approximately 40% in comparison to the production without 1-dodecanol and up to the inventors' knowledge is the best hydroxytyrosol titer reported. However, the tyrosol conversion into hydroxytyrosol was not very efficient since only 60% of tyrosol was converted into hydroxytyrosol, comparing with tyrosol strain ST191. Hydroxytyrosol production in E. coli has been reported before (0.65 g/L of hydroxytyrosol) from glucose, by engineering E. coli BW25113 with heterologous expression of ScARO10 gene, overexpression of ADH6, tyrA, ppsA, tktA and aroG genes, and knocking out feaB gene. They achieved this production by inducing cells with 0.5 mM of IPTG in M9Y medium at 37° C. Comparing this result to the one obtained in this study, Li and his team produced approximately 30% less hydroxytyrosol, which could be explained by the use of 0.5 mM of IPTG instead of 0.1 mM of IPTG, overexpressing more genes than us and knocking out only feaB gene.
Cells were grown in LB medium for 2 h, washed and resuspended in M9Y+2% of glucose+0.1 mM of IPTG (regular media)) at 30 C and incubated for 72 h. The low copy number for hpaBC favours the accumulation of hydroxytyrosol. The addition of dodecanol increased the hydroxytyrosol production in approximately 40%. The biphasic system stabilized hydroxytyrosol production. The pheaL and feaB gene knockouts and the 02 limitation decreased the hydroxytyrosol accumulation.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21155780.6 | Feb 2021 | EP | regional |
| 117340 K | Jul 2021 | PT | national |
| 21196276.6 | Sep 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/053036 | 2/8/2022 | WO |
