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Establishing an artificial pathway for efficient biosynthesis of hydroxytyrosol Xianglai Li, Zhenya Chen, Yifei Wu, Yajun Yan, Xinxiao Sun, and Qipeng Yuan ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00385 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on January 1, 2018
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ACS Synthetic Biology
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Establishing an artificial pathway for efficient biosynthesis of hydroxytyrosol
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Xianglai Lia, Zhenya Chena, Yifei Wua, Yajun Yanb, Xinxiao Suna*, Qipeng Yuana*
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a
State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China
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b
College of Engineering, The University of Georgia, Athens, GA 30602, USA
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Corresponding authors:
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*Xinxiao Sun
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[email protected] 10
Telephone: +86-10-64431557
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Address: 15 Beisanhuan East Road, Chaoyang District, Beijing 100029, China
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*Qipeng Yuan
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[email protected] 15
Telephone: +86-10-64437610; fax: +86-10-64437610;
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Address: 15 Beisanhuan East Road, Chaoyang District, Beijing 100029, China
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Abstract
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Hydroxytyrosol (HT) is a valuable natural phenolic compound with strong antioxidant
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activity and various physiological and pharmaceutical functions. In this study, we established
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an artificial pathway for HT biosynthesis. First, efficient enzymes were selected to construct
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tyrosol biosynthetic pathway. Aro10 from Saccharomyces cerevisiae was shown to be a
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better ketoacid decarboxylase than Kivd from Lactococcus lactis for tyrosol production.
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While knockout of feaB significantly decreased accumulation of the byproduct
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4-hydroxyphenylacetic acid, overexpression of alcohol dehydrogenase ADH6 further
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improved tyrosol production. The titers of tyrosol reached 1469 ± 56 mg/L from tyrosine and
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620 ± 23 mg/L from simple carbon sources, respectively. The pathway was further extended
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for HT production by overexpressing Escherichia coli native hydroxylase HpaBC. To
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enhance transamination of tyrosine to 4-hydroxyphenylpyruvate, NH4Cl was removed from
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the culture media. To decrease oxidation of HT, ascorbic acid was added to the cell culture.
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To reduce the toxicity of HT, 1-dodecanol was selected as the extractant for in situ removal of
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HT. These efforts led to an additive increase in HT titer to 1243 ± 165 mg/L in the feeding
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experiment. Assembly of the full pathway resulted in 647±35 mg/L of HT from simple
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carbon sources. This work provides a promising alternative for sustainable production of HT,
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which shows scale up potential.
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KEYWORDS: hydroxytyrosol; microbial synthesis; shikimate pathway; biphasic cultivation;
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4-hydroxyphenylacetic acid 3-hydroxylase
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Phenolic compounds constitute the biggest group of natural antioxidants. They have drawn
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much attention due to their diverse biological activities and beneficial health effects.
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Hydroxytyrosol (3, 4-dihydroxyphenylethanol or HT) is a natural phenolic compound present
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mainly in olives. It exhibits powerful antioxidant activity and contributes to the beneficial
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properties of virgin olive oil.1 Numerous studies have demonstrated the disease-preventing
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potential of HT. It can protect blood lipids from oxidative damage,2 inhibit platelet
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aggregation,3
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anti-inflammatory6 and anti-microbial activities.7 In addition, HT has good bioavailability
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and no known toxic effects.8,
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supplements, functional foods and even medicine. However, due to the lack of efficient
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production methods, this valuable compound is not commercially available in large scale.
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HT naturally exists in fruits and vegetables, but it is mostly abundant in olive trees (Olea
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europaea) as a product of oleuropein degradation. Consequently, olive tree derivatives are the
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main accessible sources for HT. Processes have been developed to extract HT from fresh
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olive leaves 10, olive pomace 11 or olive mill wastewaters (OMWWs).12 Although the starting
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materials are cheap and abundant, these processes have several drawbacks, such as use of
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strongly-acid aqueous steam, low recovery yields and long duration.13 Chemical synthesis
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processes have been developed using structure analogues of HT, such as tyrosol and
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2,3-dihydroxybenzaldehyde as the starting materials.14,
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relatively expensive and the processes often require protection and de-protection steps,
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lowering the overall yields.13 Besides chemical conversion, bioconversion studies have also
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been conducted to produce HT from precursors like tyrosol, 2-phenylethanol and
and
scavenge
free
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radicals.4
It
also
exhibits
anti-carcinogenic,5
Therefore, it possesses promising applications in food
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However, these substrates are
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3-nitrophenethyl alcohol (3NPA). In one study, mushroom tyrosinase was used to catalyze
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the hydroxylation of tyrosol to HT while ascorbic acid was used as a reducing agent to
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prevent over-oxidation of HT.16 However, tyrosinase is instable and expensive and its activity
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is inhibited by both the phenols and ascorbic acid. Some aromatic compound degrading
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microorganisms, such as Serratia marcescens, Pseudomas aeruginosa, Pseudomonas putida
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F6, Halomonas sp. strain HTB24 were used to convert tyrosol to HT by their native
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hydroxylases.17-20 4-Hydroxyphenylacetic acid 3-hydroxylase was demonstrated to be
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responsible for tyrosol hydroxylation.21 In another study, the activity of toluene
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4-monooxygenase (T4MO) was improved by a combination of directed evolution and
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rational design for HT production from 2-phenylethanol by two successive oxidation
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reactions.22 An engineered nitrobenzene dioxygenase (NBDO) was also used for HT
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production from 3NPA.23 Both T4MO and NBDO suffer from poor regio-specificity and low
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enzyme activity.
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A major obstacle to bioconversion production of HT is the cost of the substrates. The advent
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of metabolic engineering and synthetic biology provides new opportunities and great
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potential to tackle this obstacle by reconstituting natural pathways or even designing artificial
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pathways.24 In fact, efficient production of a variety of aromatic compounds, such as
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5-hydroxytryptophan25, caffeic acid26
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carbon sources using metabolic engineered microorganisms. To date, only one pathway has
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been reported for microbial production of HT.28 This pathway starts from E. coli native
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metabolite tyrosine. Tyrosine is converted to HT by the sequential catalysis of tyrosine
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hydroxylase (TH), L-Dopa decarboxylase, tyramine oxidase and native dehydrogenases. The
and flavoniods27 has been achieved from simple
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first two enzymes are from mammalians and their encoding genes were codon-optimized for
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better expression in E. coli. In addition, a cofactor regeneration pathway was constructed to
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support the activity of TH. With strain and pathway optimization, the final strain produced
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0.08 mM (12.3 mg/L) HT from glucose.28 This research opens the possibility for HT
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production from simple carbon sources. However, the pathway efficiency need to be further
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improved for economical production of HT.
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Considering the wide potential applications of HT and the lack of economical feasible
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production methods, in this study we designed a novel artificial pathway. HT was produced
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from tyrosine or from simple carbon sources using metabolic engineered E. coli and under
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optimized conditions the titers reached 1243 ± 165 mg/L and 647±35 mg/L, respectively.
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Results and Discussion
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Design an artificial pathway for HT biosynthesis
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HT is a natural product of oleuropein degradation. Previously, HT has been produced from
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tyrosol via whole cell bioconversion.21 The conversion of tyrosol to HT requires only one
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hydroxylation step and 4-HPA 3-hydroxylase (HpaBC) from P. aeruginosa was demonstrated
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to be able to carry out this reaction. HpaBC is widely distributed in many microorganisms
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including E. coli. E. coli HpaBC has broad substrate activity and has been used for
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hydroxylation of a variety of aromatic compounds, including coumaric acid,26 monolignols29
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and flavonoids30. In this study, E. coli HpaBC was selected to perform the hydroxylation of
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tyrosol to HT. We purified the enzyme and characterized its catalytic parameters toward
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tyrosol (km= 18.1 ± 0.7 µM, Kcat = 12.1 ± 0.1 min-1) following the protocol described by Lin et.al30.
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As tyrosol is a relative expensive precursor, it is necessary to extend upward to achieve HT
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production from a cheaper substrate or ultimately from simple carbon sources such as glucose
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and glycerol. To this end, an efficient tyrosol biosynthesis pathway is required to connect
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with the downstream conversion step. So far, two pathways have been reported for tyrosol
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biosynthesis. In the first pathway, tyrosine is converted to tyrosol by the consecutive catalysis
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of tyrosine decarboxylase, tyramine oxidase and alcohol dehydrogenase (ADH).31 In the
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second pathway, tyrosol, as an intermediate of salidroside biosynthesis, was produced from
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4-hydroxyphenylpyruvate (4-HPP) through the sequential catalysis of ketoacid decarboxylase
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(KDC) and ADH.32 Judging from the obtained titers of tyrosol, the efficiency of the latter
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pathway is significantly higher than that of the former one. Accordingly, an artificial pathway
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was designed for HT biosynthesis (Figure 1).
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Tyrosol production from tyrosine
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Feeding experiment was carried out to determine the optimal enzyme combination for tyrosol
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production. 4-HPP is an intermediate in tyrosine biosynthesis and E. coli native
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aminotransferases such as TyrB catalyze the reversible transformation between tyrosine and
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4-HPP. Considering the easier commercial availability, tyrosine instead of 4-HPP was chosen
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as the substrate for the bioconversion study. Two KDCs, Kivd from L. lactis and Aro10 from
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S. cerevisiae, were selected as the candidates.
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At first, E. coli BW25113 was transformed with plasmid pZE-Kivd, generating strain BK,
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and used for the feeding experiment. In 48 h, BK produced only 198 ± 9 mg/L of tyrosol, but
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accumulated 450 ± 35 mg/L of 4-HPA (Figure 2A&B). In E. coli, FeaB is the main
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dehydrogenase that converts 4-HPAA to 4-HPA. To reduce 4-HPA production, gene feaB in
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the chromosome was knocked out, generating strain BW∆feaB. BW∆feaB was transformed
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with plasmid pZE-Kivd, generating strain B∆K. Strain B∆K produced 499 ± 8 mg/L of
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tyrosol while the amount of 4-HPA was significantly decreased to 36 ± 2 mg/L. To test
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whether native ADHs are sufficient to reduce 4-HPAA to tyrosol, ADH6 with broad substrate
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ranges from S. cerevisiae was co-expressed with Kivd on plasmid pZE-Kivd-ADH6. Strain
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BKA produced 600 ± 23 mg/L of tyrosol but still accumulated 293 ± 12 mg/L of 4-HPA. In
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comparison, strain B∆KA produced 778 ± 33 mg/L of tyrosol with only 30 ± 3 mg/L of
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4-HPA (Figure 2A&B). It was observed that the cell growth decreased with the increase of
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tyrosol production (Figure 2C), which was generally coincident with the trend of the
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inhibition experiment (Figure S1). Aro10 was shown to have better activity toward
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phenylpyruvate than Kivd.33 Since 4-HPP is very similar with phenylpyruvate, we assumed
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that Aro10 may be a better candidate than KivD for tyrosol production. We then replaced
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Kivd with Aro10, generating plasmid pZE-Aro10-ADH6. In 48 h, strain B∆AA carrying this
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plasmid produced 1469 ± 56 mg/L of tyrosol along with 117 ± 13 mg/L of 4-HPA at 37 ℃.
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We also investigated the effect of cultivation temperature on the conversion efficiency. At
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30 ℃, strain B∆AA produced 692 ± 20 mg/L of tyrosol, which is less than half of that at 37 ℃
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(Figure 2D).
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From the results obtained above, we drew several conclusions. Firstly, disrupting feaB can
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significantly decrease 4-HPA accumulation, but there are still other minor aldehyde
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dehydrogenases that can act on 4-HPAA. Secondly, although disrupting feaB is sufficient to
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decrease 4-HPA production, overexpressing ADH6 is further beneficial to tyrosol production.
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Thirdly, Aro10 performs better than Kivd for tyrosol production. Fourthly, 37 ℃ is better
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than 30 ℃ for the bioconversion.
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Tyrosol production from simple carbon sources
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After achieving efficient production of tyrosol from tyrosine, we further investigated de novo
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production of tyrosol. To this end, BW∆feaB was transformed with plasmids pZE-Aro10-
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ADH6 and pCS-TPTA, generating strain B∆AAT. Plasmid pCS-TPTA is a medium copy
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plasmid overexpressing four key enzymes to boost the carbon flux through shikimate
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pathway. In shake flask experiment cell growth of B∆AAT peaked at 24 h, but tyrosol titer
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kept increasing till 48 h, reaching 550 ± 26 mg/L (Figure 3A). We also tested the capability of
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another strain QH4∆feaB for tyrosol production. Strain QH4 is a derivative of the
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phenylalanine overproducer ATCC31884 and has been successfully used for microbial
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production of a variety of aromatic compounds.26, 29, 34 Transforming the same two plasmids
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into QH4∆feaB generated strain Q∆AAT. Strain Q∆AAT produced 620 ± 23 mg/L of tyrosol
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in 48 h (Figure 3B). As for 4-HPA accumulation, strains B∆AAT and Q∆AAT showed
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different trends. During the cultivation process, B∆AAT accumulated less than 40 mg/L of
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4-HPA (Figure 3A). However, the titer of 4-HPA produced by strain Q∆AAT increased to 273
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± 6 mg/L during the first 24 hours, and then decreased to 86 ± 5 mg/L at 48 h (Figure 3B).
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HT production from tyrosine
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To extend tyrosol pathway for HT production, E. coli native hydroxylase HpaBC was
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overexpressed on plasmid pCS-HpaBC. Strain BW∆feaB was transformed with
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pZE-Aro10-ADH6 and pCS-HpaBC, generating strain B∆AAH, for bioconversion of HT
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from tyrosine. Strain B∆AAH was able to efficiently convert tyrosine to HT. HT titer peaked
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at 24 h, reaching 710 ± 34 mg/L. Unlike that of tyrosol, the titer of HT decreased
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significantly with time after 24 h. At 48 h, only 261 ± 23 mg/L of HT remained in the culture
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(Figure 4A). This indicates that introduction of an additional hydroxyl group makes HT less
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stable than tyrosol. We also observed that the color of the cell cultures turned black. This is
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because HpaBC can also act on tyrosine and the product L-Dopa is unstable and readily
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oxidized to melanin. To circumvent this problem, in our previous study a co-culture system
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was designed to prevent the accessibility of HpaBC to tyrosine.29 Here, the problem was
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alleviated by simply removing NH4Cl in the M9 medium. The removal of NH4Cl is supposed
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to enforce the cells to use tyrosine as an alternative nitrogen source, which can provide a
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driving force for transamination of tyrosine to 4-HPP. As expected, using the modified M9
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medium lacking NH4Cl HT titer peaked at 36 h and reached 831 ± 49 mg/L, although the cell
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density was decreased by 20 %. Moreover, ascorbic acid was added to the cell culture to
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decrease HT oxidation. This effort further improved the titer to 972 ± 160 mg/L (Figure 4A).
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HT showed antibacterial activity against several bacterial strains.35 To test its inhibition effect
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on E. coli BW25113, HT was added to the cell cultures immediately after inoculation to the
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final concentrations ranging from 0-2000 mg/L. The cell growth of BW25113 was
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significantly inhibited at concentrations higher than 1000 mg/L. The final OD600 at 2000
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mg/L of HT was about 40 % of that of the control (Figure 5A).
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To alleviate the toxic effect of HT, the biphasic process was used to achieve in situ removal of
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HT from the culture media. A key step to biphasic production is to choose a suitable organic
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solvent with good biocompatibility and product affinity. Ethyl acetate is the most commonly
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used solvent to extract phenolic compounds including HT from OMWWs under acidic
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conditions36. However, this solvent is highly volatile and toxic to the cells. Therefore, we
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chose hexyl acetate instead of ethyl acetate as a candidate solvent. Besides, 1-octanol has
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been used as an extractant for phenol production by solvent-tolerant host P. putida S12,
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leading to a twofold increase in phenol titer.37 In another study, 1-dodecanol was proved to be
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a promising solvent for 3-methylcatechol production in a biphasic partitioning bioreactor 38.
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As HT is also a phenolic compound, it is expected that the longer-chain alcohols could be
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suitable solvents for HT extraction.
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A limitation accompanied with organic solvents is their potential toxicity towards the
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biocatalyst. To test the toxicity of the three extractants, they were added to the cell cultures
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with the O/W ratio of 10%. It was shown that hexyl acetate and 1-octanol exhibited severe
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inhibition on cell growth while 1-dodecanol showed only minor inhibition (Figure 5B). We
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also measured the partition coefficients of HT between the extractants and the M9 medium.
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As indicated in Figure 5C, these organic solvents showed modest capability to extract HT
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from the culture medium. Although 1-octanol has the highest partition coefficient,
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1-dodecanol was used for further biphasic extraction study due to its good biocompatibility.
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For this, 25 mL of 1-dodecanol was added to 50 mL of the cell cultures at 12 h after
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inoculation. Adding 1-dodecanol, together with removal of NH4Cl led to the production of
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1037 ± 108 mg/L of HT at 36 h. By the combination of adding1-dodecanol and ascorbic acid,
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and removing NH4Cl, the final titer of HT reached 1243 ± 165 mg/L, which is 75 % higher
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than that in the original medium (Figure 4A). Besides, it is observed that the biphasic
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cultivation also led to increased accumulation of the intermediate tyrosol (Figure 4B).
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HT production from simple carbon sources
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To realize de novo production of HT, E. coli BW∆feaB and QH4∆feaB were transformed
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with plasmids pZE-Aro10-ADH6 and pCS-TPTA-HpaBC, generating strain B∆AATH and
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Q∆AATH. Strain B∆AATH produced 507±35 mg/L of HT at 32 h. Adding ascorbic acid
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improved the titer to 596±63 mg/L at 48 h. Adding both ascorbic acid and 1-dodecanol led
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to the production of 647±35 mg/L of HT at 48 h (Figure 6). Tyrosol and 4-HPA were
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accumulated only in tiny amount. However, when strain Q∆AATH was used for HT
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production, the titer reached only 395±25 mg/L at 48 h (Figure 7A). Meanwhile, both
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tyrosol and 4-HPA were observed to be accumulated in significant amount (Figure 7B&C).
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Like the previous observation, the titer of 4-HPA peaked at 24 h and decreased thereafter.
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Therefore, BW∆feaB is more suitable than QH4∆feaB for HT production.
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Discussion
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In this work, we designed a novel biosynthetic pathway and achieved efficient HT production
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from simple carbon sources. HT titer reached 647±35 mg/L, which is over 50 times higher
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than that obtained previously.28 The product titer largely relies on the performance of pathway
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enzymes. While several mammalian enzymes were involved in the previous pathway, the
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enzymes used in this work are all from microorganisms. Generally, microbial enzymes are
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more compatible with microbial hosts, because many mammalian enzymes require
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posttranslational modifications and inner membrane structures for their activity which is
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lacking in microorganisms.
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Tyrosol is a key intermediate in HT biosynthesis. Microbial production of tyrosol had been
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reported before.32,
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production and made several new observations. We noticed that disruption of feaB can
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significantly decrease but cannot completely eliminate 4-HPA accumulation especially under
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the genetic background of QH4, indicating there are other aldehyde dehydrogenases that can
39
As a key step toward HT production, we also optimized tyrosol
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oxidize 4-HPAA. Besides, in previous studies 4-HPAA was reduced to tyrosol by E. coli
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native dehydrogenases. In this work, we found that overexpression of a dehydrogenase is
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beneficial to tyrosol production. Moreover, exogenous aromatic amino acid transaminase had
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been overexpressed in E. coli to convert tyrosine to 4-HPP.39 We observed that E. coli native
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transaminase is sufficient to carry out the transamination. Eliminating NH4Cl could provide a
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driving force and enhance the transamination.
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As an antioxidant, HT is easy to be oxidized during the production process. Adding ascorbic
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acid to the culture media was shown to be beneficial for HT production. HT shows inhibition
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effect on cell growth. To circumvent this problem, biphasic cultivation was adopted for in situ
244
removal of HT. Among the three extractants selected, 1-dodecanol was shown to be the
245
optimal one with good biocompatibility and modest extraction capability. Ethyl acetate had
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been used as the solvent to extract HT from OMWWs under acidic conditions
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Acidification can decrease the dissociation of HT in the water system and improve the
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partition coefficient. However, the cell growth will be retarded under lower pH, which
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generates a dilemma between high partition coefficient and good cell growth. Even with
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modest partition coefficient, the biphasic system was able to significantly improve HT
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production. In further work, other strategies may be adopted to further alleviate the toxicity
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and facilitate the recovery of HT, e.g., introducing acyl transferase to further convert HT into
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its esters.
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Materials and methods
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Bacterial strains and culture media.
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E. coli XL1-Blue (from Stratagene) was used for plasmid construction and propagation. E.
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.
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coli BW25113 (from Coli Genetic Stock Center) and QH4 were used for production of
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tyrosol and HT. LB medium was used for seed culture. Modified M9 medium was used for
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feeding experiments and de novo production of tyrosol and HT. LB medium contains 10 g/L
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tryptone, 5 g/L yeast extract, and 10 g/L NaCl. The modified M9 medium contains 2 g/L
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MOPS (morpholinepropanesulfonic acid), 10 g/L glycerol, 2.5 g/L glucose, 6 g/L Na2HPO4,
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0.5 g/L NaCl, 3 g/L KH2PO4, 2 g/L NH4Cl, 1mM MgSO4, 0.1 mM CaCl2 and yeast extract.
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When needed, ampicillin, kanamycin and ascorbic acid were added to the medium at 100
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µg/mL, 50 µg/mL, and 1 g/L, respectively.
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DNA manipulation. Plasmids pZE12-luc (high copy), pCS27 (medium copy) were used for
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pathway assembly. Plasmids pCS-HpaBC, pCS-TPTA were constructed as described in our
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previous study.29,
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genome DNA. Gene encoding Kivd was amplified from L. lactis genome DNA. Gene Kivd
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was cloned into pZE12-luc using KpnI and XbaI, generating pZE-Kivd. Plasmid
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pZE-Kivd-ADH6 was constructed by inserting Kivd and ADH6 encoding genes into
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pZE12-luc using KpnI, PstI and XbaI. Plasmid pZE-Aro10 was constructed by inserting
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Aro10 encoding gene into pZE12-luc using KpnI and SphI. Plasmid pZE-Aro10-ADH6 was
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constructed by inserting Aro10 and ADH6 encoding genes into pZE12-luc using KpnI,
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BamHI and XbaI. Plasmid pCS-TPTA-HpaBC was constructed by inserting the expressing
275
cassette PLlacO1-HpaBC into SpeI/SacI sites of pCS-TPTA. Gene feaB on the chromosome
276
of BW25113 and QH4 was inactivated following the standard protocol of RED
277
recombination as described previously41 . Plasmids and strains used in this study are listed in
278
Table1. Primers used in this study were listed in Table S1.
40
Genes encoding Aro10 and ADH6 were amplified from S.cerevisiae
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Feeding experiments. Feeding experiments were conducted to examine the production of
280
tyrosol and HT from tyrosine. Single colonies were inoculated into 4 mL of LB media
281
containing appropriate antibiotic(s) and cultured overnight at 37 ℃. Overnight cultures (1
282
mL) were inoculated into 50 mL of M9Y media containing 5 g/L yeast extract and
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appropriate antibiotic(s). Cells were grown at 37 ℃ and induced with 0.5 mM
284
isopropyl-β-D-thiogalactoside (IPTG). The induced strains were fed with tyrosine at 0 h, 12 h
285
and 24 h after inoculation (each time at 1 g/L). Samples were taken at several time points.
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Optical densities at 600 nm (OD600) were measured for cell growth and the concentrations of
287
product and by-products were analyzed by HPLC. HT was extracted with ethyl acetate and
288
analyzed by ESI-MS and the molecular weight was accordance with that of the HT standard
289
(Figure S2).
290
De novo production of tyrosol and HT
291
For de novo production of tyrosol and HT, 1 mL overnight cultures were inoculated into 50
292
mL of M9Y media containing 2 g/L yeast extract and appropriate antibiotics. Cells were
293
cultivated at 37 °C and induced with 0.5 mM IPTG for 48 h. Samples were taken every 12 h.
294
The cell densities (OD600) were measured and the concentrations of the products and the
295
intermediates were analyzed by HPLC. For biphasic production of HT, 25 mL of 1-dodecanol
296
was added to 50 mL of the cell culture at 12 h after inoculation. Both the water phase and the
297
organic phase were subjected to HPLC analysis. The total titers were calculated by adding the
298
concentrations in the water phase with ½ of the concentrations in the organic phase.
299
Toxicity test and estimation of the partition coefficient
300
To test the toxicity of HT, HT was fed to the cell cultures of E. coli BW25113 to the final
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concentrations of 0, 250, 500, 1000 and 2000 mg/L, respectively. Similarly, to test the
302
toxicity of the organic solvents, 5 mL of the individual organic solvent was added to 50 mL of
303
the cell cultures. The toxicity was evaluated by monitoring cell growth.
304
To estimate the partition coefficient of HT, M9 medium containing 500 mg/L of HT was
305
mixed thoroughly with equal volume of the organic solvent. After ten minutes’ standing,
306
samples were taken and HPLC analysis was carried out. The partition coefficient was
307
expressed as the ratio of HT concentrations in the organic and the water phases.
308
HPLC analysis. Tyrosol, 4-hydroxyphenylacetic acid (4-HPA) and HT were all purchased
309
from Aladdin Chemical Industry and used as the standards. Both the standards and samples
310
were analyzed and quantified by HPLC (HITACHI) equipped with a reverse-phase Diamonsil
311
C18 column (Diamonsil 5 µm, 250 × 4.6 mm) and UV–VIS detector. Solvent A was methanol
312
and solvent B was water with 0.1% trifluoroacetic acid. The column temperature was set to
313
28℃. The following gradient was used at a flow rate of 1 mL/min: 90 % to 40 % solvent B
314
for 18 min, 40 % to 90 % solvent B for 1 min, and 90 % solvent B for an additional 6 min.
315
Quantification were based on the peak areas at specific wavelengths (276 nm for tyrosol, 240
316
nm for 4-HPA and 280 nm for HT).
317 318
Author information
319
Corresponding authors
320
Email:
[email protected]. Phone: +86-10-64431557
321
Email:
[email protected]. Phone: +86-10-64437610
322
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Author Contributions
324
XL, XS and QY conceived the study and wrote the manuscript. XL, ZC, and YW performed
325
the experiments. XS, QY and YY directed the research and revised the manuscript.
326 327
Notes: A patent application about this technology has been filed by Beijing University of
328
Chemical Technology.
329 330
Acknowledgments
331
The authors would like to acknowledge financial support of the National Natural Science
332
Foundation of China (21606012, 21636001 and 21776008) and the Fundamental Research
333
Funds for the Central Universities (buctrc201613).
334 335
Supporting Information. Table S1 showing primers used in this study; Figure S1 showing
336
cell growth of E. coli BW25113 at different concentrations of tyrosol; Figure S2 showing
337
ESI-MS results of hydroxytyrosol.
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Table 1. Plasmids and strains used in this study Plasmids
Feature
Source
pZE12-luc
PLlacO1, ColE ori, luc, Ampr
Ref. 36
r
pCS27
PLlacO1, P15A ori, Kan
Ref. 36
pZE-Kivd
pZE12-luc, Kivd from L. lactis
This study
pZE-Kivd-ADH6
pZE12-luc, Kivd from L. lactis and ADH6 from S. cerevisiae
This study
pZE-Aro10
pZE12-luc, ARO10 from S. cerevisiae
This study
pZE-Aro10-ADH6
pZE12-luc, ARO10 and ADH6
This study
pCS-TPTA
pCS27, tyrA, ppsA, tktA, aroGfbr from E. coli
Ref. 26
pCS-HpaBC
pCS27, hpaBC from E. coli
Ref. 26
pCS-TPTA-HpaBC
pCS27, PLlacO1-TPTA and PLlacO1-HpaBC
This study
Strains
Genotype
Source
BW25113
rrnBT14 ∆lacZWJ16 hsdR514 ∆araBADAH33 ∆rhaBADLD78
Coli Genetic Stock Center
QH4
E. coli ATCC 31884 with pheA and tyrA disrupted
Ref. 23
BW∆feaB
BW25113 with feaB disrupted
This study
QH4∆feaB
QH4 with feaB disrupted
This study
BK
BW25113 carrying plasmid pZE-Kivd
This study
B∆K
BW∆feaB carrying plasmid pZE-Kivd
This study
BKA
BW25113 carrying plasmid pZE-Kivd-ADH6
This study
B∆KA
BW∆feaB carrying plasmid pZE-Kivd-ADH6
This study
B∆AA
BW∆feaB carrying plasmid pZE-Aro10-ADH6
This study
B∆AAT
BW∆feaB carrying plasmids pZE-Aro10-ADH6 and pCS-TPTA
This study
Q∆AAT
QH4∆feaB carrying plasmid pZE-Aro10-ADH6 and pCS-TPTA
This study
B∆AAH
BW∆feaB carrying plasmid pZE-Aro10-ADH6 and pCS-HpaBC
This study
B∆AATH
BW∆feaB carrying plasmid pZE-Aro10-ADH6 and pCS-TPTA
This study
Q∆AATH
QH4∆feaB carrying plasmid pZE-Aro10-ADH6 and pCS-TPTA
-HpaBC
-HpaBC
441 442
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443
Figure captions
444 445 446 447 448 449 450 451
Figure 1. The novel biosynthetic pathway of hydroxytyrosol. Compounds: 4-HPP, 4-hydroxyphenylpyruvate; 4-HPAA, 4-hydroxyphenylacetaldehyde; 4-HPA, 4-hydroxyphenylacetic acid. Enzymes: KDC, ketoacid decarboxylase; ADH, alcohol dehydrogenase; HpaBC, 4-hydroxyphenylacetic acid 3-hydroxylase; TyrB, aromatic-amino-acid aminotransferase; FeaB, phenylacetaldehyde dehydrogenase. Dashed arrows indicate that the branches are blocked.
452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471
Figure 2. Tyrosol production from tyrosine using different strains. Five strains were used and their genetic details are shown in Table 1. (A) Cell growth. (B) Tyrosol production. (C) 4-HPA accumulation. (D) Tyrosol production using strain B∆AA at 37 ℃ and 30 ℃. Error bars represent standard deviations of three replicates. Figure 3. De novo production of tyrosol by (A) strain B∆AAT and (B) strain Q∆AAT. Refer to Table 1 for the genetic details. Error bars represent standard deviations of three replicates. Figure 4. Optimization of bioconversion conditions for HT production from tyrosine. Strain BAAH was used for the bioconversion experiment. (A) Profiles of HT accumulation at different conditions. (B) Profiles of tyrosol accumulation at different conditions. Error bars represent standard deviations of three replicates.
Figure 5. Selection of an appropriate extractant for biphasic production of HT. (A) Effect of HT on cell frowth. (B) Effect of the extractants on cell growth. (C) Partition coefficients of HT between the extractants and the M9 medium. Error bars represent standard deviations of three replicates.
Figure 6. De novo production of HT. Strain B∆AATH was used for the experiments. Error bars represent standard deviations of three replicates.
472 473 474 475 476
Figure 7. De novo production of HT. Strain Q∆AATH was used for the experiments. (A) HT production. (B) Tyrosol accumulation. (C) 4-HPA accumulation. Error bars represent standard deviations of three replicates.
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Figure 1. The novel biosynthetic pathway of hydroxytyrosol. Compounds: 4-HPP, 4-hydroxyphenylpyruvate; 4-HPAA, 4-hydroxyphenylacetaldehyde; 4-HPA, 4-hydroxyphenylacetic acid. Enzymes: KDC, ketoacid decarboxylase; ADH, alcohol dehydrogenase; HpaBC, 4-hydroxyphenylacetic acid 3-hydroxylase; TyrB, aromatic-amino-acid aminotransferase; FeaB, phenylacetaldehyde dehydrogenase. Dashed arrows indicate that the branches are blocked. 94x45mm (300 x 300 DPI)
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Figure 2. Tyrosol production from tyrosine using different strains. Five strains were used and their genetic details are shown in Table 1. (A) Cell growth. (B) Tyrosol production. (C) 4-HPA accumulation. (D) Tyrosol production using strain B∆AA at 37 ℃ and 30 ℃. Error bars represent standard deviations of three replicates.
208x156mm (300 x 300 DPI)
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Figure 3. De novo production of tyrosol by (A) strain B∆AAT and (B) strain Q∆AAT. Refer to Table 1 for the genetic details. Error bars represent standard deviations of three replicates. 241x324mm (300 x 300 DPI)
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Figure 4. Optimization of bioconversion conditions for HT production from tyrosine. Strain BAAH was used for the bioconversion experiment. (A) Profiles of HT accumulation at different conditions. (B) Profiles of tyrosol accumulation at different conditions. Error bars represent standard deviations of three replicates. 224x292mm (300 x 300 DPI)
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Figure 5. Selection of an appropriate extractant for biphasic production of HT. (A) Effect of HT on cell frowth. (B) Effect of the extractants on cell growth. (C) Partition coefficients of HT between the extractants and the M9 medium. Error bars represent standard deviations of three replicates. 898x245mm (300 x 300 DPI)
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Figure 6. De novo production of HT. Strain B∆AATH was used for the experiments. Error bars represent standard deviations of three replicates. 209x148mm (300 x 300 DPI)
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Figure 7. De novo production of HT. Strain Q∆AATH was used for the experiments. (A) HT production. (B) Tyrosol accumulation. (C) 4-HPA accumulation. Error bars represent standard deviations of three replicates. 582x153mm (300 x 300 DPI)
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