Metabolic Engineering of Saccharomyces cerevisiae for High-Level

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Biotechnology and Biological Transformations

Metabolic Engineering of Saccharomyces cerevisiae for High-level Production of Salidroside from Glucose Jingjie Jiang, Hua Yin, shuai wang, Yibin Zhuang, Shaowei Liu, Tao Liu, and Yanhe Ma J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01272 • Publication Date (Web): 19 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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Journal of Agricultural and Food Chemistry

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Metabolic Engineering of Saccharomyces cerevisiae for High-level Production of

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Salidroside from Glucose

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Jingjie Jiang,† Hua Yin,‡,§ Shuai Wang,‡,⊥ Yibin Zhuang,‡,§ Shaowei Liu,*,† Tao Liu, *,‡,§ Yanhe

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Ma ‡,§

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†

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University of Science and Technology, Shanghai 200237, China

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‡

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China

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§

College of Biotechnology, the State Key Laboratory of Bioreactor Engineering, East China

Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308,

Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin

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300308, China

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⊥

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Technology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin

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300457, China

National and Local United Engineering Laboratory of Metabolic Control Fermentation

14 15 16 17 18 19

ACS Paragon Plus Environment

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ABSTRACT: Salidroside is an important plant-derived aromatic compound with diverse

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biological properties. Due to inadequate natural resources, the supply of salidroside is currently

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limited. In this work, we engineered the production of salidroside in yeast. First, the aromatic

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aldehyde synthase (AAS) from Petroselinum crispum was overexpressed in Saccharomyces

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cerevisiae when combined with endogenous Ehrlich pathway to produce tyrosol from tyrosine.

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Glucosyltransferases from different resources were tested for ideal production of salidroside in

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the yeast. Metabolic flux was enhanced towards tyrosine biosynthesis by overexpressing

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pathway genes and eliminating feedback inhibition. The pathway genes were integrated into

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yeast chromosome, leading to a recombinant strain that produced 239.5 mg/L salidroside and

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965.4 mg/L tyrosol. The production of salidroside and tyrosol reached up to 732.5 mg/L and

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1394.6 mg/L, respectively, by fed-batch fermentation. Our work provides an alternative way for

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industrial large-scale production of salidroside and tyrosol from S. cerevisiae.

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KEYWORDS: Saccharomyces cerevisiae, salidroside, tyrosol, glucosyltransferase

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INTRODUCTION

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Rhodiola is a highly valued edible herb, which has been well-known as “Tibetan Ginseng” with a

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long history. It is widely used as the health food to bolster immunity, memory, and learning,

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scavenge superoxide radicals, as well as relieve altitude sickness.1 The plants of the Rhodiola

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genus prefer to grow in cold areas and at high altitude such as the slopes of Hengduan mountains

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and the Himalayas.2 Salidroside [2-(4-hydroxyphenyl)ethyl-β-D-glucopyranoside] is the main

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bioactive ingredient of the plant Rhodiola. It possesses various biological properties including

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anti-aging, anoxia resistance, anti-inflammation activities as well as cardiovascular disease

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prevention, anti-cancer, nerve, and brain cell protection.3-8 Salidroside has attracted much

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attention as potential nutraceutical supplements. Tyrosol, the aglycone of salidroside, has been

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proven to reduce the risk of Alzheimer’s diseases scientifically.9 In addition, it is commonly used

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as a food additive of the bitter ingredient to improve the taste of Japanese sake.10

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Three different methods have been used to produce salidroside including direct extraction

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from plants or cell tissue cultures,11,12 chemical synthesis as well as microbial synthesis.13-16

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Direct extraction method has been limited due to inadequate natural resources and low content of

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plant tissues, and chemical synthesis has disadvantages of employing toxic chemicals and

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complex procedures. Microbial production may provide an alternative way to meet the

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commercial demand of salidroside.

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Three pathways have been reported for the aglycone tyrosol formation. The first one is known

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as Ehrlich pathway in yeast wherein 4-hydroxyphenylpyruvate was decarboxylated into the

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corresponding aldehyde followed by the action of endogenous alcohol dehydrogenases

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(ADHs).17 In the second pathway, tyrosine decarboxylase and tyramine oxidase transform


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tyrosine into the corresponding aldehyde, which was then converted into tyrosol by the activity

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of ADHs.10 More recently, aromatic aldehyde synthase from P. crispum or Rhodiola was

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identified as the enzyme that catalyzes the direct conversion of tyrosine to 4-

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hydroxyphenylacetaldehyde, ultimately forming tyrosol.14,16 For the first time, we achieved the

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production of salidroside in microorganisms by introducing pyruvate decarboxylase ARO10

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from the Ehrlich pathway and glucosyltransferase UGT73B6 into E. coli.15 After that, the

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production of salidroside was also achieved by using the aromatic aldehyde synthase from P.

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crispum and glucosyltransferase AtUGT85A1 in E. coli.14

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S. cerevisiae is a user-friendly heterologous host that has been used to produce plant

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secondary metabolites. As the heterologous host for producing valuable chemicals, yeast offers

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various advantages such as consistent quality, rapid growth rate, simple culture conditions, and

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simple extraction procedures.18 Furthermore, it serves as a food-grade strain for producing health

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beneficial products that are widely applied in food, cosmetic and pharmaceutical fields.

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Recently, the biosynthetic pathway of salidroside has been fully elucidated, and salidroside was

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produced in yeast for the first time. However, the titer of salidroside produced by the strain was

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low.16

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Hence, in this study, we aimed to engineer S. cerevisiae to overproduce salidroside. Firstly, we

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heterologously overexpressed aromatic acetaldehyde synthase from P. crispum into yeast in

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combination with endogenous Ehrlich pathway to enhance the production of tyrosol.

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Subsequently, the UDP-glycosyltransferases (UGTs) from different resources were screened to

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produce glucosylated tyrosol. Furthermore, we optimized the biosynthetic pathway of tyrosine to

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increase the carbon flux towards tyrosol formation. Finally, for stable overexpression, the genes

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in the pathway were integrated into yeast chromosome through homologous recombination. To


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our knowledge, this is the first time to construct a plasmid-free strain for overproducing

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salidroside, and a total of 732.5 mg/L salidroside, as well as 1394.6 mg/L tyrosol, were produced

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through fed-batch fermentation in a 5 L bioreactor.

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MATERIALS AND METHODS

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Plasmids, Strains, and Medium. All the plasmids and strains used in this study are listed in

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Table 1 and Table 2. S. cerevisiae BY4742 (MATα his3∆1 leu2∆0 lys2∆0 ura3∆0) was used as a

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parent strain for genetic engineering in this study. Engineered yeast strains were cultured in

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synthetic complete medium (SC) without uracil or leucine where appropriate. Escherichia coli

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DH5α was used for plasmid construction. Yeast transformation of plasmids or linearized DNA

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fragments was carried out with lithium acetate/single-stranded carrier DNA/PEG method.19 The

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chemicals used in this study to produce salidroside were purchased from Sigma-Aldrich.

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Plasmid Construction. All the primers involved in this study are listed in Table S1. PcAAS

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(GenBank: AAA33860.1) from Petroselinum crispum, AtUGT73C5 (GenBank: NM_129235.4)

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and AtUGT85A1 (GenBank: NM_102089) from Arabidopsis thaliana, RsUGT73B6 (GenBank:

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AY547304) and RsUGT74R1 (GenBank: EF508689.1) from Rhodiola sachalinensis were

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synthesized by Generay Biotech Co., Ltd. (Shanghai, China). The DNA fragments with

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promoters PTEF1, PPGK1, PTDH3 and terminator TTEF1 were amplified individually by PCR from

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genomic DNA of S. cerevisiae using primers PTEF1-F/PTEF1-R, PTDH3-F/PTDH3-R, PPGK1-F/PPGK1-R

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or TTEF1-F/TTEF1-R. DNA fragments of promoter PTEF1 and terminator TTEF1 were assembled by

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overlapping PCR with primers PTEF1-F/TTEF1-R, resulting in the PTEF1-TTEF1 fragment. The

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amplified product was then cloned into NheI and HindIII sites of plasmid pESC-URA3, leading

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to the plasmid pCf300. DNA fragments of promoters PPGK1 and PTDH3 were used as templates


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with primer pair PTDH3-F/PPGK1-R for amplifying the PPGK1-PTDH3 fragment containing promoter

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PPGK1 and terminator PTDH3 by PCR. The amplified fragment was digested and cloned into the

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BamHI and SpeI sites of plasmid pCf300, yielding the plasmid pCf301. The DNA fragment was

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digested from the plasmid pCf301 with SpeI and NheI and then cloned into pESC-LEU2,

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resulting in the plasmid pCf302 (Figure S1). The PcAASsyn fragment was amplified by PCR with

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primer pair PcAASsyn-F/PcAASsyn-R, digested and inserted into pCf302 via SalI/HindIII,

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resulting in the plasmid pCf302-PcAASsyn. The RsUGT73B6syn, RsUGT74R1syn, AtUGT73C5syn,

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and AtUGT85A1syn were amplified with primer pairs RsUGT73B6syn-F/RsUGT73B6syn-R,

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RsUGT74R1syn-F/RsUGT74R1syn-R, AtUGT73C5syn-F/AtUGT73C5syn-R, or AtUGT85A1syn-

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F/AtUGT85A1syn-R, digested and ligated into pCf302 through SpeI/BglII, resulting in the

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plasmids pCf302-RsUGT73B6syn, pCf302-RsUGT74R1syn, pCf302-AtUGT73C5syn or pCf302-

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AtUGT85A1syn, respectively. AtUGT85A1syn gene was also cloned into pCf302-PcAASsyn,

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resulting in the plasmid pCf302-PcAASsyn-AtUGT85A1syn. The genes ARO4 and ARO7 were

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amplified from genomic DNA of S. cerevisiae. Two mutant genes, ARO4K229L, and ARO7G141S

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were generated by directed mutagenesis method.20 ARO4K229L was amplified by PCR using

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primers

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SalI/HindIII, resulting in the plasmid pCf301-ARO4K229L. ARO7G141S gene was amplified using

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primers ARO7G141S-Up-F/ARO7G141S-Down-R, digested and cloned into pCf301-ARO4K229L

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through SpeI/BglII, resulting in the plasmid pCf301-ARO4K229L-ARO7G141S. AROL fragment was

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amplified by PCR with primers AROL-F/AROL-R from genomic DNA of E. coli, digested and

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cloned into pCf301-ARO4K229L-ARO7G141S by restriction sites AatII and NheI, resulting in the

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plasmid pCf301-ARO4K229L-ARO7G141S-AROL.

ARO4K229L-Up-F/ARO4K229L-Down-R,

digested

and

cloned

into

pCf301

via


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Modular Integration into Yeast Chromosome. Cassettes with genes encoding a URA3

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selectable marker, ARO4K229L, ARO7G141S, and AROL were integrated into YPRC∆15 DNA site

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of S. cerevisiae chromosome X. Two fragments were amplified from pCf301-ARO4K229L-

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ARO7G141S-AROL using primer pairs YPRC∆15-UP-URA3-F/URA3-R and TADH1-F/TCYC1-

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YPRC∆15-Down-R and then transformed into strain BY4742. The resulting colonies were

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selected on the SC plates without uracil and verified by PCR amplification and DNA sequencing.

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The resulting strain was designated as ST-010 and used for further engineering. PcAASsyn and

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AtUGT85A1syn were integrated into YJR056C DNA site of strain ST-010. DNA fragment

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containing PPGK1-AtUGT85A1syn-TADH-PTDH3-PcAASsyn-TTEF1 and a LEU2 selectable marker

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was amplified by PCR from the plasmid pCf302-PcAASsyn-AtUGT85A1syn with primer pairs

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YJR056C-UP-LEU2-F/LEU2-R and TADH1-F/TTEF1-YJR056C-Down-R. The amplified products

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were transformed into strain ST-010, and the resulting colonies were selected on the SC plates

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without uracil and leucine. The colonies were verified by PCR amplification and DNA

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sequencing, and the colony having the highest salidroside titer was designated as ST-011.

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Cultivation Conditions of the Recombinant S. cerevisiae strains. The recombinant yeasts

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were specifically described in Figure 2. The wild-type yeast strain was cultivated in

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yeast extract peptone dextrose (YPD) medium containing 10 g/L yeast extract, 20 g/L peptone,

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20 g/L glucose, and all the engineered strains were cultured in SC medium containing 6.7 g/L

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yeast nitrogen base, 2 g/L dropout mix, and 20 g/L glucose as the sole carbon source. 1 mL

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overnight culture of recombinant S. cerevisiae was diluted into 50 mL fresh SC media to give an

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OD600 equal to 0.2 and grew at 30 °C and 200 rpm for 120 h or 216 h (for strain ST-011). For

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feeding experiments, the strains were cultured in SC media with 2 mM tyrosine (for strains ST-

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001 and ST-002) or 2 mM tyrosol (for strains ST-003, ST-004, ST-005, and ST-006).


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Fed-batch Fermentation of Strain ST-011. Strain ST-011 was used to produce salidroside

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and tyrosol in a 5-L bioreactor. The strain was cultivated in 50 mL SC media without leucine and

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uracil in 250 mL shaking flasks at 30 °C, with 200 rpm for 24 h. Then the seed was transferred

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into the 5-L bioreactor containing 2.5 L SC fermentation broth to give an OD600 equal to 0.2. The

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temperature was maintained at 30 °C, and the agitation speed was set at 300 rpm. The pH was

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maintained at 5.5 through the automatic addition of 5 M ammonia water. Air flow rate was kept

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at 4 L/min. 500 g/L glucose solution was fed into the medium continuously to maintain the

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concentration of glucose at 10-20 g/L in the medium.

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Analysis and Identification of Tyrosol and Salidroside. High-performance liquid

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chromatography-mass spectrometry (HPLC-MS) was performed on an Agilent 1260 system with

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1260 Infinity UV detector and a BrukermicroTOF-QII mass spectrometer with an ESI ionization

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probe. The column used was AgelaInnoval C18 (4.6 × 250 mm) with a 5 µm particle size. The

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HPLC conditions were as follows: solvent A = 0.1% methanolic acid in H2O; solvent B =

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methanol; flow rate = 1 mL/min; 0-25 min 80% A and 20% B; 26-40 min 80% A and 20% B to

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100% B (linear gradient); 41-45 min 100% B. The supernatant of fermentation broth, tyrosol,

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and salidroside (purchased from Sigma-Aldrich) were submitted for HPLC-MS analysis and

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detected at UV = 224 nm. To quantify the amount of tyrosol and salidroside, standard calibration

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curves were prepared by using known concentrations of the standard compounds dissolved in

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culture medium. R2 value for the standard curve was greater than 0.999. All the experiments

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were carried out in triplicate and repeated at least twice. The titers were presented as means ±

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SD.

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RESULTS


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Synthesis of Tyrosol in Recombinant S. cerevisiae. Tyrosol is a native metabolite of yeast

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that is derived from endogenous Ehrlich pathway, and we introduced PcAAS to enhance the

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production of tyrosol in yeast (Figure 1). The fermentation broth of the engineered strains was

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analyzed by HPLC-MS with tyrosol as the positive control. Analysis of the metabolic profile of

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the strain ST-001 harboring empty plasmid pCf302 revealed a peak with a retention time of 12.5

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min in the HPLC chromatogram, which is identical with that of tyrosol standard (Figure 3). The

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compound corresponding to the peak has a molecular ion at 121.3586 ([M - H2O + H]+), which

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was identical with that of tyrosol standard. Thus, we confirmed the production of tyrosol by the

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control strain, and the highest titer of tyrosol produced by strain ST-001 was 170.8 ± 3.5 mg/L.

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Strain ST-002 carrying pCf302-PcAASsyn was cultured in SC medium without leucine. The titer

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of tyrosol was increased to 401.6 ± 9.8 mg/L in 120 h. When 2 mM tyrosine was incubated with

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the strains ST-001 or ST-002, the production of tyrosol was improved to a titer of 398.9 ± 6.5

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mg/L or 610.9 ± 10.2 mg/L, respectively (Figure 3).

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Glucosylation of Tyrosol to Salidroside in Recombinant S. cerevisiae. Genes

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RsUGT73B6syn, RsUGT74R1syn, AtUGT73C5syn, and AtUGT85A1syn were individually introduced

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into S. cerevisiae, resulting in strains ST-003, ST-004, ST-005, and ST-006, respectively. 2 mM

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tyrosol was added to the fermentation broth. HPLC analyses of the metabolites produced by

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recombinant strains ST-005 carrying AtUGT73C5syn and ST-006 harboring AtUGT85A1syn

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displayed a new peak at retention time of 10 min (Figure 4). The new compound has a molecular

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ion at m/z 301.1331 ([M + H]+), which was identical with that of salidroside. The titers of

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salidroside produced by strains ST-005 and ST-006 were 58.4 ± 5.8 mg/L and 224.5 ± 3.2 mg/L

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in 120 h, respectively. HPLC analyses of culture broth of strains ST-002 carrying RsUG73B6syn

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and ST-005 containing AtUGT73C5syn revealed a new peak with retention time of 6.5 min. The


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new metabolite has a molecular ion at m/z 323.1096 ([M + NH4]+), which was identical with that

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of icariside D2 characterized in our previous work.15

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De novo Production of Salidroside in yeast. The gene encoding AtUGT85A1syn was

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introduced into the strain ST-002, generating strain ST-007. The recombinant strain ST-007

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carrying pCf302-PcAASsyn-AtUGT85A1syn was incubated in SC medium with the strain ST-001

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as the control. After cultivation for 120 h, the production of salidroside and tyrosol were

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analyzed by HPLC (Figure 5A). The titers of salidroside and tyrosol reached 79.8 ± 5.6 mg/L

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and 257.5 ± 3.2 mg/L, respectively. To increase the supply of tyrosine in the cell, the plasmid

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containing pCf301-ARO4K229L-ARO7G141S-AROL was transferred into the strain ST-007

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harboring pCf302-PcAASsyn-AtUGT85A1syn, which resulted in a new strain ST-009. The

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recombinant strains ST-009 containing pCf301-ARO4K229L-ARO7G141S-AROL and pCf302-

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PcAASsyn-AtUGT85A1syn was cultivated in SC medium for producing salidroside. The analysis

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of the strain ST-009 culture supernatant using HPLC revealed two peaks corresponding to

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salidroside and tyrosol (Figure 5B). The titers of salidroside and tyrosol reached 107.1 ± 4.2

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mg/L and 434.7 ± 9.4 mg/L, respectively.

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Construction of Plasmid-free Salidroside Overproducing Strain. To establish a genetically

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stable strain, genes ARO4K229L, ARO7G141S, AROL, PcAASsyn, and AtUGT85A1syn were integrated

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into yeast chromosome in two steps. Genes including ARO4K229L, ARO7G141S, AROL, and a

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URA3 selection marker were integrated at the YPRC∆15 site in the S. cerevisiae chromosome

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XVI via one-step homologous recombination (Figure S2), generating series of tyrosol producing

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strains. The highest titer of tyrosol reached up to 399.2 ± 5.6 mg/L in 120 h (Figure S4).

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Sequentially, the genes including AtUGT85A1syn, PcAASsyn, and a LEU2 selective marker were

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integrated at YJR056C DNA site in the S. cerevisiae chromosome X (Figure S3). This


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subsequently resulted in salidroside production, where the titers varied from 51.1 ± 2.5 mg/L to

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239.5 ± 5.8 mg/L in 216 h (Figure S5). The strain having the highest titer of salidroside was

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designated as ST-011. Tyrosol produced by strain ST-011 was analyzed by HPLC (Figure 5C),

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and the titer of tyrosol reached 965.4 ± 12.4 mg/L.

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Production of Salidroside by Fed-batch Fermentation. To further improve salidroside

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production, strain ST-011 was cultured through fed-batch fermentation in a 5-L bioreactor. As

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the carbon resource, glucose was added continuously at 10 g/L/h to keep the concentration

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constantly between 10-20 g/L. After cultivation for 72 h, cell growth reached stationary phase

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with OD600 = 7.5. Production of salidroside continued to improve until 120 h. After that,

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salidroside yield remained stable with a titer of 732.5 ± 4.9 mg/L. The production of tyrosol

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reached up to 1394.6 ± 12.8 mg/L (Figure 6).

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DISCUSSION

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In this study, we metabolically engineered S. cerevisiae for high-level production of

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salidroside from glucose, and the highest titer of salidroside reached 732.5 mg/L by fed-batch

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fermentation. As far as we know, it was the highest titer of de novo production of salidroside

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reported in microorganisms. The strain also produced 1394.6 mg/L tyrosol.

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A set of studies have been carried out to synthesize tyrosol and salidroside from glucose in E.

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coli and S. cerevisiae.14-16 Pyruvate decarboxylase ARO10 converted 4-hydroxypyruvate acid to

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4-hydroxybenzaldehyde via Ehrlich pathway in S. cerevisiae.17 Several studies have

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demonstrated that aromatic aldehyde synthase can directly convert tyrosine to 4-

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hydroxybenzaldehyde.14,16,21-22 In order to increase the production of the aglycone tyrosol,

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PcAASsyn was overexpressed to synthesize tyrosol from tyrosine, resulting in the successful


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improvement of the production of tyrosol. Our work suggested that a combination of PcAASsyn

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and ARO10 was more helpful for tyrosol production by a possible synergistic effect.

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We determined whether the precursor is adequate for the production of the aglycone tyrosol by

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feeding experiments. The quantity of tyrosol produced by the strain ST-002 was improved by

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supplying exogenous tyrosine, indicating that the tyrosine is one of the limiting factors for

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tyrosol production. It is widely reported that ARO4 and ARO7 are feedback-controlled enzymes

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that limit the production of tyrosine. Alleviating feedback inhibition via two mutagenesis of

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ARO4K229L and ARO7G141S enabled a 200-fold increase of the extracellular aromatic amino acids

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concentration in yeast.23 The study conducted by Angelica Rodriguez et al. showed that

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overexpressing shikimate kinase AROL enhanced the metabolic flux of intermediate compound

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to p-coumaric acid.24 Ultimately, integration of genes ARO4K229L, ARO7G141S, and AROL into

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chromosome led to an approximately 2-fold improvement of tyrosol production, compared to

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that produced by the wild strain.

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Glycosyltransferases are mainly responsible for the synthesis of glycosides. Salidroside is one

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of the important bioactive substances produced by the plant Rhodiola. The native UGT from

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Rhodiola was not efficient to convert tyrosol to salidroside.16 We screened several substituted

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UGTs from other plants, which were characterized according to their bioactivities towards the

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production of tyrosol including RsUGT73B6syn, RsUGT74R1syn, AtUGT73C5syn, and

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AtUGT85A1syn.14-15,25 Our previous work has demonstrated that RsUGT73B6syn converted

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tyrosol into salidroside in E. coli.15 However, RsUGT73B6syn catalyzed only the formation of

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trace amounts of icariside D2 in our yeast strain, and no salidroside was produced. This may be

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caused by the low expression or unsuitable modification of the glucosyltransferase by the host S.

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cerevisiae. The exact reason needs to be further investigated in the future if needed. We tested


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AtUGT73C5syn and AtUGT85A1syn from A. thaliana for glycosylation of tyrosol. According to

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the bioconversion experiments, AtUGT85A1syn acts as a more appropriate biocatalyst for the

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production of salidroside in yeast. However, a significant amount of tyrosol was not converted to

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salidroside, indicating that glucosylation is still the limiting step for salidroside formation. In the

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future, more efforts should be devoted to acquiring UGTs with higher activity by screening from

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nature or by protein engineering. Besides, further optimization of the UDP-glucose pathway will

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be helpful for enhancing the production of salidroside in the coming works.

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For stable expression, we integrated all the pathway genes of salidroside biosynthesis into the

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yeast genome. More than 400 δ sequences were dispersed throughout the yeast genome, and each

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site has the potential for integration via yeast homologous recombination. Genes integrated into

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some δ sites were reported to have higher transcriptional levels, whereas certain sites are

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available for multiple copies of stable integration at a time.26-27 Moreover, the δ-mediated

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integrations demonstrated no significant influences on cell growth and expression of cloned

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genes.26 In this study, YPRC∆15 and YJR056C DNA sites were selected as locations for genes

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integration, as reported in the biosynthesis of β-carotene, ginsenosides, and geranylgeraniol.27-29

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The production of salidroside was increased from 79.8 mg/L to 239.5 mg/L. To further increase

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the production of salidroside, a fed-batch fermentation was developed for the strain ST-011 in a

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5-liter bioreactor.

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Salidroside has great potential to be used in food and pharmaceutical fields due to its diverse

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biological activities and low toxicity. Currently, salidroside production mainly depends on the

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direct extraction from wild Rhodiola. However, tough living environment, slow growth, and

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over-harvesting are pushing the wild Rhodiola to extinction. Field cultivation of Rhodiola is


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costly, time-consuming, and thus is inapplicable.30 Herein, the engineered yeast strain may offer

288

an alternative way for industrial large-scale production of salidroside.

289

ASSOCIATED CONTENT

290

Supporting Information

291

The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

292

Detailed schematic diagrams for the relative plasmids construction; Schematic diagrams for a

293

one-step method of gene integration; Genes integration into chromosome for tyrosol or

294

salidroside production; Primers and DNA sequences applied in this study.

295

AUTHOR INFORMATION

296

Corresponding Author

297

*Phone: +86-22-24828718. E-mail: [email protected]; [email protected].

298

Funding

299

This work was supported by grants from the National Natural Science Foundation of China (no.

300

31770104, no. 21302214 and no. 31400026), and the Biological Resources Service Plan of CAS

301

(no. ZSTH-023).

302

Notes

303

The authors declare no competing financial interest.

304

ABBREVIATIONS USED

305

AAS, aromatic aldehyde synthase; ADH, alcohol dehydrogenase; ARO4, 3-deoxy-D-arabino-

306

heptulosonate-7-phosphate (DAHP) synthase; ARO7, chorismite mutase; AROL, shikimate


14

Page 15 of 26


307

kinase; UGT, UDP-glucosyltransferase; PCR, polymerase chain reaction; OD600, optical density

308

at 600 nm; HPLC-MS, high-performance liquid chromatography-mass spectrometry.

309

REFERENCES

310

(1) Yang, Y.; Liu, Z.; Feng, Z.; Jiang, J.; Zhang, P. Lignans from the root of Rhodiola crenulata. J

311

Agr Food Chem 2012, 60, 964-72.

312

(2) Lei, Y.; Gao, H.; Tsering, T.; Shi, S.; Zhong, Y. Determination of genetic variation in

313

Rhodiola crenulata from the Hengduan Mountains region, China using inter-simple sequence

314

repeats. Gene Mol Biol 2006, 29, 1275-1277.

315

(3) Diaz-Lanza, A.; Recuero-Carretero, C.; Villaescusa-Castillo, L.; Silvan-Sen, A.; Bermejo-

316

Benito, P. Lignan and phenylpropanoid glycosides from Phillyrea latifolia and their in vitro anti-

317

inflammatory activity. Planta Med 2001, 67, 219-223.

318

(4) Bonanome, A.; Pagnan, A.; Caruso, D.; Toia, A.; Xamin, A.; Fedeli, E.; Berra, B.;

319

Zamburlini, A.; Ursini, F.; Galli, G. Evidence of postprandial absorption of olive oil phenols in

320

humans. Nutr Metab Cardiovas 2000, 10, 111.

321

(5) JinPing, Z.; AnHeng, L.; RongRong, H.; Juan, Z.; Xin, J.; WeiFeng, J.; JianZong, C.

322

Salidroside protects cardiomyocyte against hypoxia-induced death: a HIF-1α-activated and

323

VEGF-mediated pathway. Eur J pharmacol 2009, 607, 6.

324

(6) Udintsev, S. N.; Schakhov, V. P. Decrease of cyclophosphamide haematotoxicity by Rhodiola

325

rosea root extract in mice with Ehrlich and Lewis transplantable tumors. Eur J Cancer 1991, 27,

326

1182.

327

(7) Zhang, L.; Yu, H.; Sun, Y.; Lin, X.; Chen, B.; Tan, C.; Cao, G.; Wang, Z. Protective effects of

328

salidroside on hydrogen peroxide-induced apoptosis in SH-SY5Y human neuroblastoma cells.

329

Eu J Pharmacol 2007, 564, 18-25.


15


Page 16 of 26

330

(8) Zhang, L.; Yu, H.; Zhao, X.; Lin, X.; Tan, C.; Cao, G.; Wang, Z. Neuroprotective effects of

331

salidroside against beta-amyloid-induced oxidative stress in SH-SY5Y human neuroblastoma

332

cells. Neurochem Int 2010, 57, 547-55.

333

(9) St-Laurent-Thibault, C.; Arseneault, M.; Longpré, F.; Ramassamy, C. Tyrosol and

334

hydroxytyrosol, two main components of olive oil, protect N2a cells against amyloid-β-induced

335

toxicity. Involvement of the NF-κB signaling. Cur Alzheimer Res 2011, 8, -.

336

(10) Satoh, Y.; Tajima, K.; Munekata, M.; Keasling, J. D.; Lee, T. S. Engineering of a tyrosol-

337

producing pathway, utilizing simple sugar and the central metabolic tyrosine, in Escherichia coli.

338

J Agr Food Chem 2012, 60, 979-84.

339

(11) Xu, J. Z.; Feng, P. S. Activity of tyrosol glucosyltransferase and improved salidroside

340

production through biotransformation of tyrosol in Rhodiola sachalinensis cell cultures. J

341

Biotechnol 1998, 61, 69-73.

342

(12) Wu, S.; Zu, Y.; Wu, M. High yield production of salidroside in the suspension culture of

343

Rhodiola sachalinensis. J Biotechnol 2003, 106, 33-43.

344

(13) Zhang, S.; Shang, G.; Li, Z.; Wang, A.; Cai, M. A New Approach to synthesis of salidroside.

345

Chinese J Chem 1997, 48, 2881-2885.

346

(14) Chung, D.; Kim, S. Y.; Ahn, J. H. Production of three phenylethanoids, tyrosol,

347

hydroxytyrosol, and salidroside, using plant genes expressing in Escherichia coli. Sci Rep 2017,

348

7.

349

(15) Bai, Y.; Bi, H.; Zhuang, Y.; Liu, C.; Cai, T.; Liu, X.; Zhang, X.; Liu, T.; Ma, Y. Production of

350

salidroside in metabolically engineered Escherichia coli. Sci Rep 2014, 4, 6640.

351

(16) Torrens-Spence, M. P.; Pluskal, T.; Li, F.-S.; Carballo, V.; Weng, J.-K. Complete pathway

352

elucidation and heterologous reconstitution of Rhodiola salidroside biosynthesis. Mol Plant


16

Page 17 of 26


353

2018, 11, 205-217.

354

(17) Hazelwood, L. A.; Daran, J. M.; Maris, A. J. A. V.; Pronk, J. T.; Dickinson, J. R. The Ehrlich

355

Pathway for Fusel Alcohol Production: a Century of research on Saccharomyces cerevisiae

356

metabolism. Appl Environ Microb 2008, 74, 2259-2266.

357

(18) Borodina, I.; Nielsen, J. Advances in metabolic engineering of yeast Saccharomyces

358

cerevisiae for production of chemicals. Biotechnol J 2014, 9, 609-620.

359

(19) Gietz, R. D.; Schiestl, R. H. Frozen competent yeast cells that can be transformed with high

360

efficiency using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2007, 2, 1-4.

361

(20) Zheng, L.; Baumann, U.; Reymond, J. L. An efficient one-step site-directed and site-

362

saturation mutagenesis protocol. Nucleic Acids Res 2004, 32, e115.

363

(21) Kaminaga, Y.; Schnepp, J.; Peel, G.; Kish, C. M.; Bennissan, G.; Weiss, D.; Orlova, I.;

364

Lavie, O.; Rhodes, D.; Wood, K. Plant phenylacetaldehyde synthase is a bifunctional

365

homotetrameric enzyme that catalyzes phenylalanine decarboxylation and oxidation. J Bio Chem

366

2006, 281, 23357-66.

367

(22) Torrens-Spence, M. P.; Gillaspy, G.; Zhao, B.; Harich, K.; White, R. H.; Li, J. Biochemical

368

evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde

369

synthase enzyme. Biochem Bioph Res Co 2012, 418, 211-6.

370

(23) Luttik, M. A.; Vuralhan, Z.; Suir, E.; Braus, G. H.; Pronk, J. T.; Daran, J. M. Alleviation of

371

feedback inhibition in Saccharomyces cerevisiae aromatic amino acid biosynthesis:

372

quantification of metabolic impact. Metab Eng 2008, 10, 141-153.

373

(24) Rodriguez, A.; Kildegaard, K. R.; Li, M.; Borodina, I.; Nielsen, J. Establishment of a yeast

374

platform strain for production of p-coumaric acid through metabolic engineering of aromatic

375

amino acid biosynthesis. Metab Eng 2015, 31, 181-188.


17


Page 18 of 26

376

(25) Yu, H. S.; Ma, L. Q.; Zhang, J. X.; Shi, G. L.; Hu, Y. H.; Wang, Y. N. Characterization of

377

glycosyltransferases responsible for salidroside biosynthesis in Rhodiola sachalinensis.

378

Phytochem 2011, 72, 862-870.

379

(26) Lee, F. W. F.; Silva, N. A. D. Sequential δ Integration for the regulated insertion of cloned

380

genes in Saccharomyces cerevisiae. Biotechnol Progr 1997, 13, 368.

381

(27) Li, S.; Ding, W.; Zhang, X.; Jiang, H.; Bi, C. Development of a modularized two-step (M2S)

382

chromosome integration technique for integration of multiple transcription units in

383

Saccharomyces cerevisiae. Biotechnol Biofuels 2016, 9, 232.

384

(28) Song, T. Q.; Ding, M. Z.; Zhai, F.; Liu, D.; Liu, H.; Xiao, W. H.; Yuan, Y. J. Engineering

385

Saccharomyces cerevisiae for geranylgeraniol overproduction by combinatorial design. Sci Rep

386

2017, 7.

387

(29)

388

Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides. Metab Eng

389

2013, 20, 146.

390

(30) Zhou, W.; Bi, H.; Zhuang, Y.; He, Q.; Yin, H.; Liu, T.; Ma, Y. Production of cinnamyl

391

alcohol glucoside from glucose in Escherichia coli. J Agr Food Chem 2017, 65, 2129.

392

FIGURE CAPTIONS

393

Figure 1. Biosynthetic pathway for the production of salidroside in S. cerevisiae. PEP:

394

phosphoenolpyruvate; E4P: erythrose-4-phosphate; DAHP: 3-deoxy-D-arabino-heptulosonic

395

acid 7-phosphate, DHQ: 3-dehydroquinate, DHS: 3-dehydro-shikimate, SHP: shikimate-3-

396

phosphate, EP3P: 5-enolpyruvylshikimate-3-phosphate. The enzymes overexpressed in the

397

salidroside pathway are shown in red.

398

Figure 2. Recombinant yeasts harboring modules for overexpressing various enzymes.

Dai, Z.; Liu, Y.; Zhang, X.; Shi, M.; Wang, B.; Wang, D.; Huang, L.; Zhang, X.,


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399

Figure 3. (A) HPLC analysis of standard tyrosol (I) and products in the fermentation supernatant

400

of the recombinant strains ST-001 harboring empty vector pCf302 (as control), ST-002

401

harboring pCf302-PcAASsyn. (B) Mass analysis of tyrosol (I) from fermentation supernatant of

402

strain ST-002. (C) The influence of 2 mM tyrosine (fed in the fermentation broth) on the tyrosol

403

production.

404

Figure 4. (A) HPLC analysis of tyrosol (I) and its glucosides (II) and (III) in the fermentation

405

supernatant of the strains ST-003 harboring pCf302-RsUGT73B6syn, ST-004 harboring pCf302-

406

RsUGT74R1syn, ST-005 harboring pCf302-AtUGT73C5syn, ST-006 harboring pCf302-

407

AtUGT85A1syn. (B) and (C) Mass analysis of salidroside (II) and icariside D2 (III), respectively.

408

Figure 5. (A) HPLC analysis of tyrosol (I) and salidroside (II) in the fermentation supernatant of

409

strains ST-001 harboring empty vector pCf302 (as control), ST-007 harboring pCf302-

410

PcAASsyn-AtUGT85A1syn. (B) HPLC analysis of tyrosol (I) and salidroside (II) in the

411

fermentation supernatant of the strains ST-008 harboring empty vectors pCf301 & pCf302 (as

412

control), strain ST-009 harboring pCf301-ARO4K229L-ARO7G141S-AROL & pCf302-PcAASsyn-

413

AtUGT85A1syn. (C) HPLC analysis of tyrosol (I) and salidroside (II) in the fermentation

414

supernatant of recombinant strain ST-011

415

Figure 6. Time profiles of cell density, titers of salidroside and tyrosol production by strain ST-

416

011 through fed-batch fermentation for 168 h.

417 418 419


19


Page 20 of 26

420 421

TABLES

422

Table 1 Plasmids used in this work Plasmids

Descriptions

Sources

pESC-URA3

URA3; 2 µm ori with PGAL1, PPGK10; Amp

Novagen

pESC-LEU2

LEU2; 2 µm ori with PGAL1, PPGK10; Amp

Novagen

pCf301

URA3; 2 µm ori with PPGK1, PTDH3, PTEF1;

This study

Amp pCf302

LEU2; 2 µm ori with PPGK1, PTDH3, PTEF1;

This study

Amp pCf302-PcAASsyn

pCf302 carrying PTDH3-PcAASsyn-TTEF1

This study

cassette pCf302-RsUGT73B6syn

pCf302 carrying PPGK1-RsUGT73B6syn-

This study

TADH1 cassette pCf302-RsUGT74R1syn

pCf302 carrying PPGK1-RsUGT74R1syn-

This study

TADH1 cassette pCf302-AtUGT73C5syn

pCf302 carrying PPGK1-AtUGT73C5syn-

This study

TADH1 cassette pCf302-AtUGT85A1syn


This study

TADH1 cassette pCf302-PcAASsynAtUGT85A1

syn


This study

syn

TADH1-PTDH3-PcAAS -TTEF1 cassette


20

Page 21 of 26


pPCf301-ARO4K229L-

pCf301 carrying PPGK1-ARO7G141S-

ARO7G141S-AROL

TADH1-PTDH3-ARO4K229L-TTEF1-PTEF1-

This study

AroL-TCYC1 cassette 423 424

Table 2 Strains used in this work Strains

Descriptions

Sources

BY4742

MATα; his3∆1; leu2∆0; lys2∆0; ura3∆0

Novagen

ST-001

BY4742 with pCf302

This study

ST-002

BY4742 with pCf302-PcAASsyn

This study

ST-003

BY4742 with pCf302-RsUGT73B6syn

This study

ST-004

BY4742 with pCf302-RsUGT74R1syn

This study

ST-005

BY4742 with pCf302-AtUGT73C5syn

This study

ST-006

BY4742 with pCf302-AtUGT85A1syn

This study

ST-007

BY4742 with pCf302-PcAASsyn-AtUGT85A1syn

This study

ST-008

BY4742 with pCf301 & pCf302

This study

ST-009

BY4742

with

pCf301-ARO4K229L-ARO7G141S-

This study

AROL & pCf302-PcAASsyn-AtUGT85A1syn ST-010

BY4742 integrating with URA3 selectable marker and

This study

PPGK1-ARO7G141S-TADH1-PTDH3-ARO4K229L-

TTEF1-PTEF1-AroL-TCYC1 cassettes into YPRC∆15 DNA site ST-011

ST-010 integrating with LEU2 selectable marker

This study

and PPGK1-AtUGT73C5syn-TADH1-PTDH3-PcAASsyn-


21


Page 22 of 26

TTEF1 cassettes into YJR056C DNA site 425 426 427

Figure 1.

428 429 430 431 432 433 434 435 436


22

Page 23 of 26


437 438 439 440

Figure 2.

441


23


Page 24 of 26

442 443 444 445

Figure 3.

446 447

Figure 4.


24

Page 25 of 26


448 449 450 451

Figure 5.

452 453 454

Figure 6.


25


Page 26 of 26

455 456 457 458 459 460 461 462

GRAPHIC FOR TABLE OF CONTENTS

463 464


26

Metabolic Engineering of Saccharomyces cerevisiae for High-Level

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