identification and microbial production of the raspberry ...identiﬁcation and microbial production...

Identification and Microbial Production of the RaspberryPhenol Salidroside that Is Active againstHuntington’s Disease1

Nicolai Kallscheuer,a,2 Regina Menezes,b,c,2 Alexandre Foito,d Marcelo Henriques da Silva ,e

Adelaide Braga,f,g Wijbrand Dekker,h David Méndez Sevillano,e Rita Rosado-Ramos,b,c Carolina Jardim,b,c

Joana Oliveira,f,g Patrícia Ferreira,f,g Isabel Rocha,f,g Ana Rita Silva,f,g Márcio Sousa,f,g

J. William Allwood,d Michael Bott,a,i Nuno Faria,f,g Derek Stewart,d,j Marcel Ottens,e Michael Naesby,h

Cláudia Nunes dos Santos,b,c,3,4 and Jan Marienhagena,i,3,4

aInstitut für Bio- und Geowissenschaften (IBG-1: Biotechnologie), Forschungszentrum Jülich, Jülich 52428,GermanybInstituto de Biologia Experimental e Tecnológica (iBET), 2781-901 Oeiras, PortugalcInstituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras,PortugaldThe James Hutton Institute, Invergowrie, DD2 5DA Dundee, Scotland, United KingdomeDepartment of Biotechnology, Delft University of Technology, 2629 HZ Delft, The NetherlandsfBiotempo, 4805-017 Guimarães, PortugalgCentre of Biological Engineering, University of Minho, 4710-057 Braga, PortugalhEvolva, 4053 Reinach, SwitzerlandiBioeconomy Science Center (BioSC), Forschungszentrum Jülich, Jülich D-52425, GermanyjSchool of Engineering and Physical Sciences, Institute of Mechanical, Process and Energy Engineering,Heriot-Watt University, Edinburgh, Scotland, United Kingdom

ORCID IDs: 0000-0003-4925-6923 (N.K.); 0000-0003-0552-8480 (R.M.); 0000-0001-6111-5256 (A.F.); 0000-0002-8606-5598 (M.H.daS.);0000-0001-7432-0726 (A.B.); 0000-0003-0643-6705 (D.M.S.); 0000-0002-1294-9227 (R.R.); 0000-0001-6929-6469 (C.J.); 0000-0001-9494-3410 (I.R.);0000-0001-6433-2694 (J.W.A.); 0000-0002-4701-8254 (M.B.); 0000-0001-5271-8178 (N.F.); 0000-0001-9838-4265 (D.S.); 0000-0001-9422-3077 (M.N.);0000-0002-5809-1924 (C.N.dosS.); 0000-0001-5513-3730 (J.M.).

Edible berries are considered to be among nature’s treasure chests as they contain a large number of (poly)phenols withpotentially health-promoting properties. However, as berries contain complex (poly)phenol mixtures, it is challenging toassociate any interesting pharmacological activity with a single compound. Thus, identification of pharmacologicallyinteresting phenols requires systematic analyses of berry extracts. Here, raspberry (Rubus idaeus, var Prestige) extracts weresystematically analyzed to identify bioactive compounds against pathological processes of neurodegenerative diseases. Berryextracts were tested on different Saccharomyces cerevisiae strains expressing disease proteins associated with Alzheimer’s,Parkinson’s, or Huntington’s disease, or amyotrophic lateral sclerosis. After identifying bioactivity against Huntington’sdisease, the extract was fractionated and the obtained fractions were tested in the yeast model, which revealed thatsalidroside, a glycosylated phenol, displayed significant bioactivity. Subsequently, a metabolic route to salidroside wasreconstructed in S. cerevisiae and Corynebacterium glutamicum. The best-performing S. cerevisiae strain was capable ofproducing 2.1 mM (640 mg L21) salidroside from Glc in shake flasks, whereas an engineered C. glutamicum strain couldefficiently convert the precursor tyrosol to salidroside, accumulating up to 32 mM (9,700 mg L21) salidroside in bioreactorcultivations (yield: 0.81 mol mol21). Targeted yeast assays verified that salidroside produced by both organisms has thesame positive effects as salidroside of natural origin.

Berries are both an enjoyable and important part of abalanced diet providing energy, nutrients and dietaryfiber, and contain a large number of compoundsshowing diverse health-promoting activities in humans(Seeram, 2012; Nile and Park, 2014). Edible berry spe-cies contain a diverse and concentrated portfolio of(poly)phenols such as anthocyanins, flavanols (epi-catechin), and stilbenes (resveratrol), which are knownto confer protection against oxidative stress and to havecytoprotective activities (Rodriguez-Mateos et al., 2014;

Bensalem et al., 2015). In recent years, there is increas-ing evidence that natural phenols in berry fruits alsoshow protective effects against metabolic disorderssuch as diabetes and obesity and against neurodegen-erative diseases (ND; Yang and Kortesniemi, 2015;Tsuda, 2016; Rosado-Ramos et al., 2018). NDs comprisea heterogeneous group of debilitating disorders causedby the progressive loss of neuronal cells. The cell typeaffected by the neurodegenerative process determinesthe appearance of specific pathologies. Frequently, the

Plant Physiology�, March 2019, Vol. 179, pp. 969–985, www.plantphysiol.org � 2019 American Society of Plant Biologists. All Rights Reserved. 969

https://plantphysiol.orgDownloaded on March 7, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

http://orcid.org/0000-0003-4925-6923

http://orcid.org/0000-0003-4925-6923

http://orcid.org/0000-0003-0552-8480

http://orcid.org/0000-0003-0552-8480

http://orcid.org/0000-0001-6111-5256

http://orcid.org/0000-0001-6111-5256

http://orcid.org/0000-0002-8606-5598

http://orcid.org/0000-0002-8606-5598

http://orcid.org/0000-0001-7432-0726

http://orcid.org/0000-0001-7432-0726

http://orcid.org/0000-0003-0643-6705

http://orcid.org/0000-0003-0643-6705

http://orcid.org/0000-0002-1294-9227

http://orcid.org/0000-0002-1294-9227

http://orcid.org/0000-0001-6929-6469

http://orcid.org/0000-0001-6929-6469

http://orcid.org/0000-0001-9494-3410

http://orcid.org/0000-0001-9494-3410

http://orcid.org/0000-0001-6433-2694

http://orcid.org/0000-0001-6433-2694

http://orcid.org/0000-0002-4701-8254

http://orcid.org/0000-0002-4701-8254

http://orcid.org/0000-0001-5271-8178

http://orcid.org/0000-0001-5271-8178

http://orcid.org/0000-0001-9838-4265

http://orcid.org/0000-0001-9838-4265

http://orcid.org/0000-0001-9422-3077

http://orcid.org/0000-0001-9422-3077

http://orcid.org/0000-0002-5809-1924

http://orcid.org/0000-0002-5809-1924

http://orcid.org/0000-0001-5513-3730

http://orcid.org/0000-0001-5513-3730

http://orcid.org/0000-0003-4925-6923

http://orcid.org/0000-0003-0552-8480

http://orcid.org/0000-0001-6111-5256

http://orcid.org/0000-0002-8606-5598

http://orcid.org/0000-0001-7432-0726

http://orcid.org/0000-0003-0643-6705

http://orcid.org/0000-0002-1294-9227

http://orcid.org/0000-0001-6929-6469

http://orcid.org/0000-0001-9494-3410

http://orcid.org/0000-0001-6433-2694

http://orcid.org/0000-0002-4701-8254

http://orcid.org/0000-0001-5271-8178

http://orcid.org/0000-0001-9838-4265

http://orcid.org/0000-0001-9422-3077

http://orcid.org/0000-0002-5809-1924

http://orcid.org/0000-0001-5513-3730

http://crossmark.crossref.org/dialog/?doi=10.1104/pp.18.01074&domain=pdf&date_stamp=2019-02-20

https://plantphysiol.org

loss of specific neuron populations is associated withthe aggregation and/or formation of amyloid struc-tures enriched with particular proteins. Alzheimer’sdisease (AD), Parkinson’s disease (PD), Huntington’sdiseases (HD), and amyotrophic lateral sclerosis (ALS),some of the major NDs, are characterized by the ag-gregation of Ab-42, a-synuclein (a-Syn), huntingtin(HTT), and FUS (an RNA-binding protein), respec-tively, representing the hallmarks of these diseases(Mason and Giorgini, 2011; Lashuel et al., 2013; Masters

et al., 2015; Zarei et al., 2015). Currently, there is no curefor NDs and therefore there is a strong interest infinding ways to treat these diseases. However, (poly)phenols have been shown to attenuate the deleteriouseffects of protein aggregation in NDs, rendering thisgroup of natural products a promising source of com-pounds for ND treatment (Rosado-Ramos et al., 2018).

Typically, (poly)phenol concentrations in the natu-rally producing plant are quite low, which means thatother strategies for a large-scale production have to bedeveloped once a pharmacologically interesting com-pound has been identified. The microbial production ofplant (poly)phenols has emerged as a true alternative todirect extraction from plants or (total) chemical syn-thesis (Milke et al., 2018). In particular, Saccharomycescerevisiae, but also bacterial host systems such as Esch-erichia coli or Corynebacterium glutamicum, have beenextensively engineered employing modern concepts ofsynthetic biology to produce plant (poly)phenols suchas resveratrol, naringenin, quercetin, and calistephin inthe last years (Lim et al., 2011; Koopman et al., 2012;Jones et al., 2017; Kallscheuer et al., 2017).

In this study, we followed a bioprospecting approachanalyzing berry extracts from Rubus idaeus (var Pres-tige) as a representative species of Rubus genus toidentify either novel compounds or known compoundswith yet uncharacterized bioactivities in connectionwith NDs. This genus includes, e.g. raspberries andblackberries, which were placed at the top ranks ofmore than 1,000 polyphenol-rich foods consumed in theUnited States (Halvorsen et al., 2006). And we havebeenworkingwith 15 species/varieties of this genus forits phytochemical composition and bioactivity (Dudniket al., 2018). This multidisciplinary approach with ex-perts from the fields of plant biology, cellular biology,microbiology, industrial biotechnology, and analyticalchemistry covered the complete workflow from thepreparation and fractionation of berry extracts, com-pound identification, and bioactivity testing to the re-construction of metabolic pathways for identifiedcompounds in microorganisms, production in biore-actors, and downstream processing.

RESULTS

Bioactivity Screening of R. idaeus (var Prestige) Extracts

Our study is based on the screening of R. idaeus (varPrestige) (poly)phenol-enriched extract for bioactivitiesagainst the cytotoxic effects of protein aggregation as-sociatedwithmajor NDs, particularly AD, PD, HD, andALS. Extracts were prepared as described in the“Material and Methods” section and tested for bioac-tivity using a yeast-based screening platform. The highdegree of evolutionary conservation of fundamentalbiological processes among eukaryotes makes yeast avaluable model organism to identify lead moleculeswith health-promoting potential against NDs (Menezeset al., 2015; Oliveira et al., 2017). Our platform includes

1This work was supported by the European Union FrameworkProgram 7 “BacHBerry” (www.bachberry.eu), (Project No. FP7-613793). iNOVA4Health - UID/Multi/04462/2013, a program finan-cially supported by Fundação para a Ciência e Tecnologia/Ministérioda Educação e Ciência, through national funds and co-funded byFEDER under the PT2020 Partnership Agreement is acknowledged.This work was also supported by Fundação para a Ciência e Tecno-logia (SFRH/BD/116597/2016 to R.R-R and IF/01097/2013 toC.N.d.S.). A.B., P.F., J.O., A.R.S., N.F., M.S., and I.R. thank the Por-tuguese Foundation for Science and Technology under the scope ofthe strategic funding of UID/BIO/04469 unit, COMPETE 2020(POCI-01-0145-FEDER-006684) and BioTecNorte operation(NORTE-01-0145-FEDER-000004) funded by the European RegionalDevelopment Fund under the scope of Norte2020 - Programa Oper-acional Regional do Norte. M.B. and J.M. acknowledge the financialsupport of the BOOST fund PNP-EXPRESS kindly funded by theBioeconomy Science Center (BioSC). The scientific activities of theBioSC were financially supported by the Ministry of Innovation, Sci-ence and Research of the German State of North Rhine-Westphaliawithin the framework of the NRW Strategieprojekt BioSC (No. 313/323-400-00213). J.M. would also like to thank the BMBF-funded pro-ject “BioLiSy” (Bioeconomic Lignan Synthesis, funding code031A554) for financial support. D.M.S., A.F., and J.A.W. acknowledgeadditional part funding from the Rural & Environment Science &Analytical Services Division of the Scottish Government.

2These authors contributed equally to this article.3Senior authors.4Author for contact: [email protected] authors responsible for distribution of materials integral to the

findings presented in this article in accordance with the Journal policydescribed in the Instructions for Authors (www.plantphysiol.org)are: Cláudia Nunes dos Santos ([email protected]) and Jan Marien-hagen ([email protected]).

A.F. sampled and extracted berry material and performed liquidchromatography-mass spectrometry (LC-MS) experiments and com-pound identification; J.W.A. developed the targeted LC-MS/MS frag-mentation method; D.M.S. oversaw the chemo-analytical work plans,developed the fractionation protocol, and performed the fractiona-tion experiments; R.M. conceived the original screening and the re-search plans and supervised the experiments on extract/fraction/compound bioactivity; R.R.-R. and C.J. performed the experimentson extract/fraction/compound bioactivity and analyzed the data;M.S. developed and performed the downstream purification; M.O.oversaw the fractionation and purification work; N.K. engineered theC. glutamicum cell factories; W.D. and M.N. constructed the salidro-side-producing yeast; A.B., N.F., and I.R. conceived and planned thebioreactor experiments with engineered C. glutamicum cell factories;A.B., N.F., J.O., P.F., A.R.S., and M.H.d.S. performed the bioreac-tor experiments with C. glutamicum cell factories; M.O., D.S., M.B.,C.N.d.S., and J.M. helped in drafting the original BacHBerry-project;and C.N.d.S. and J.M. conceived the project and wrote the article withcontributions from all authors.

www.plantphysiol.org/cgi/doi/10.1104/pp.18.01074

970 Plant Physiol. Vol. 179, 2019

Discovery and Microbial Production of Salidroside


http://www.plantphysiol.org

mailto:[email protected]

mailto:[email protected]

http://www.plantphysiol.org/cgi/doi/10.1104/pp.18.01074


yeast strains heterologously expressing genes encodingAb-42, a-Syn, HTTpQ103 (HTT 17 N-terminal aminoacids fused to 103 Gln residues), or FUS to assess bio-activity of R. idaeus (var Prestige) (poly)phenol-enriched extracts modulating pathological pathwaysassociated with AD, PD, HD, and ALS, respectively(Outeiro and Lindquist, 2003; Willingham et al., 2003;Bharadwaj et al., 2010; Ju et al., 2011). The genes codingfor the disease proteins are translationally fused to thegreen fluorescent protein (GFP) and the expression isunder control of the Gal-inducible GAL1 promoter(Flick and Johnston, 1990).In the model systems tested, R. idaeus (var Prestige)

extract was shown to be most potent in overcoming thedeleterious effects of HTTpQ103 expression (Fig. 1).Therefore, the identified R. idaeus (var Prestige) bioac-tivity against HD was further characterized. Uponshifting to a Gal medium, growth of HTTpQ103-GFP-expressing cells was impaired in comparison to that ofthe control strain, as revealed by growth curve analysisand phenotypic growth assays (Fig. 2, A and B). Theestimation of growth curve parameters using nonlinearparametric regressions (with 95% confidence intervals)indicated that expression of HTTpQ103-GFP decreasesthe final cell biomass, which was recovered by treat-ment with R. idaeus (var Prestige) (poly)phenol-enriched extract (Fig. 2A, right panel). Interestingly, R.idaeus (var Prestige)-mediated protection was efficienteven when the extract was applied as a pretreatmentbefore inducing HTTpQ103 expression with Gal(Fig. 2B). It is important to note that treatment of cellswith a similar extract from R. idaeus (var Octavia) didnot result in an observable cellular protection, indicat-ing that a R. idaeus (var Prestige)-specific compoundconfers protection against HTTpQ103-GFP cytotoxicity(Fig. 2B, last spots in the right panel). The R. idaeus (varOctavia) extract was subsequently used as a negativecontrol in further experiments. The investigation of R.idaeus (var Prestige) mode of action revealed thatgrowth improvement mediated by the extract was notassociated with any detectable difference in HTTpQ103protein levels and subcellular distribution of the inclu-sions, as revealed by immunoblotting, flow cytometry,

and fluorescence microscopy (Fig. 2, C–E). Idebenone, asynthetic analog of ubiquinone with a similar structureto (poly)phenols, known to reduce oxidative stress as-sociated with neurodegenerative complications, wasalso unable to modulate these processes. We subse-quently tested the ability of R. idaeus (var Prestige)phenolics to overcome HTTpQ103-GFP proteotoxicityby modulating its clearance. As shown in Figure 2F,HTTpQ103-GFP levels decrease at similar rates with orwithout extract incubation, indicating that this is notthe modus operandi of R. idaeus (var Prestige) phenolics.Oxidative stress is a hallmark shared by NDs, and

HTT-induced oxidative damage has been linked to celldeath (Goswami et al., 2006; Niedzielska et al., 2016;Van Raamsdonk et al., 2017). Our data show thatHTTpQ103-GFP in yeast mimics redox imbalance ob-served inHDdisease context (Browne et al., 1997, 1999),as indicated by the increase of superoxide anion pro-duction monitored by flow cytometry using the dihy-droethidium (DHE) probe (Fig. 2G). R. idaeus (varPrestige), but not R. idaeus (var Octavia), reduced DHEsignals to levels comparable to that of the control strain,indicating its potential to restore redox homeostasisimpaired by expression of HTTpQ103-GFP.

Identification of Lead Compounds in Bioactive Fractions

To identify the bioactive(s) compound(s) conferringprotection toHD, theR. idaeus (var Prestige) extract wasfractionated and 11 individual fractions (Fig. 3) weretested in the HDmodel as it has been done for the (poly)phenol-enriched extract. The growth assays revealedthat fractions 5 and 7 still conferred protection to theHDmodel, suggesting that they contain the compound(s) conferring protection against HD. Alternatively, thebioactivity of the fractions may result from the syner-gistic effect of two or more compounds.A total of 47 metabolites were found in fractions 5 (21

metabolites) and 7 (26 metabolites; Table 1) and thesecould be tentatively annotated by utilizing accuratemass, retention time, and MS/MS results acquired byliquid chromatography-mass spectrometry (LC-MS) in

Figure 1. R. idaeus (var Prestige) bioactivity againstpathological processes of four major NDs.. Yeastmodels recapitulating Ab-42, a-Syn, FUS, orHTTpQ103 proteotoxicity, respectively, were pre-grown in SC raffinose medium and growth curveswere obtained after 24 h incubation in SC Gal me-dium supplemented or not with R. idaeus (var Pres-tige) (poly)phenol-enriched extracts. The area underthe curve calculated from the growth curve is shown.The values represent the mean 6 SE of at least threebiological replicates. Statistical differences aredenoted as **P , 0.01 and ***P , 0.001, relative tothat in the control. AD, Alzheimer’s disease; ALS,amyotrophic lateral ; HD, Huntington�s disease; PD,Parkinson’s disease.

Plant Physiol. Vol. 179, 2019 971

Kallscheuer et al.



Figure 2. R. idaeus (var Prestige) bioactivity against HD is specific for this species and associatedwith the reduction of superoxideradicals. S. cerevisiaeW303-1A cells either expressingHTTpQ103-GFPor harboring the empty vector (control) were pregrown inSC raffinose medium. A, Growth curves of cells incubated in SC Gal medium for 24 h. The confidence interval for the final cell





both electrospray ionization (ESI) positive and negativemodes (Supplemental Table 1). To refine the number oflead molecules with potential protective effects on HD,the levels of compounds with tentative annotations inextracts of R. idaeus (var Prestige) were compared withthat in 14 available Rubus species, which were tested inparallel, but that did not show any bioactivity towardHD (see Methods in Supplemental Material). Thecompounds with the highest relative abundance in R.idaeus (var Prestige) in comparison with that in otherspecies or varieties were selected as tentative leadmolecules (Supplemental Fig. S1). This reduced thenumber of lead molecules from 47 to five compoundsthat comprised glycosylated derivatives of hydrox-ybenzoates, phenylethanoids, or phenylpropanoids,namely a methoxybenzoyl hexoside, hydroxyphenyl-acetate glucoside, salidroside, and two benzoyl di-hexoside isomers (Supplemental Fig. S1). Whereastentative annotations were provided based on themass spectra only, definite annotation of salidroside(Supplmental Figs. S2 and S3) was confirmed against ananalytical standard, which is only present in fraction 5.R. idaeus (var Prestige) contained the highest amount ofsalidroside compared to that in the other 14 analyzedRubus species (Fig. 4). Interestingly, salidroside hasbeen already suggested to confer a broad neuro-protective activity (Wang et al., 2015; Gao et al., 2016).Consequently, salidroside was selected for furtherexperiments.To confirm that salidroside indeed provides protec-

tion against HTTpQ103-GFP cytotoxicity, commer-cially available salidroside was also tested in the HDyeast model. Similar to the original R. idaeus (varPrestige) (poly)phenol-enriched extract, commercialsalidroside improved growth of cells expressingHTTpQ103-GFP, particularly affecting the maximumgrowth rate of cells and final biomass of the cultures(Fig. 5A). The pretreatment with salidroside alsoprotected cells from HTTpQ103-GFP cytotoxicity asrevealed by the phenotypic growth assays (Fig. 5B). Thedata show that the mechanism by which the R. idaeus(var Prestige) (poly)phenol-enriched extract mediatescellular protection is associated with the removal of

superoxide anions accumulated by the expression ofHTTpQ103-GFP (Fig. 2G). In this scenario, we expectedthat mitochondrial functionality was affected (Indoet al., 2015) and wanted to evaluate if salidroside hadthe potential to improve cell growth again. Salidrosidewas therefore supplemented under nonfermentablegrowth conditions in the presence of glycerol as the solecarbon source, which requires full mitochondrial ac-tivity for cell viability. Under this growth conditions,salidroside pretreatment (80 mM) reversed the “sensi-tive” phenotype caused byHTTpQ103-GFP expression,indicating that it may prevent mitochondrial dysfunc-tion. Remarkably, salidroside reduced superoxide an-ion production in all concentrations tested, which hintsat a great potential for restoring redox homeostasisimpaired by HTTpQ103-GFP (Fig. 5C). To test thespecificity of salidroside bioactivity for HTTpQ103-GFP-induced cell damage, we evaluated the effect ofsalidroside on growth of control yeast cells (with noHTTpQ103-GFP expression) subjected to H2O2 as wellon SOD1-defective mutants, which is characterized bydefects in cellular antioxidant responses. SOD1 is thegene encoding for the yeast superoxide dismutase1 (Rodrigues-Pousada et al., 2005), which is respon-sible for detoxifying superoxide yielding H2O2 anddioxygen. Salidroside had no effect on cells subjectedto external oxidative stress or on conditions of in-trinsic oxidative stress (Fig. 5D), underlining the as-sumed specificity toward HTTpQ103-GFP-inducedcell damage.Salidroside was found in moderate levels (approxi-

mately 20 mg kg21 fresh weight) in R. idaeus (varPrestige) berries collected in this study. No previousdata were available regarding the effects of “environ-ment” (E) and “genotype by environment” (G3E) onthe abundance levels of this compound. Although thereis potential to extract salidroside from red raspberries, ithas to be ensured that salidroside levels are consistentacross seasons and plant growth locations and thatextraction methods are available to purify salidrosidefrom a matrix rich in structurally closely related (poly)phenolics. When also considering seasonality of berryproduction, in particular the impact of climate change

Figure 2. (Continued.)biomass is shown in the right panel. B, Representative panel of drop-serial dilutions assay, phenotypic growth assays on SC Glcand SC Gal media with and without R. idaeus (var Prestige) and R. idaeus (var Octavia) extract incubation. C and D,HTTpQ103-GFP gene expression after 18 h induction in SC Gal mediumwith and without R. idaeus (var Prestige) and R. idaeus (var Octavia)extract incubation was determined by: (C) immunoblotting and respective densitometry (below) with phosphoglycerate kinase asloading control and (D) flow cytometry, GFP fluorescence versus side scatter (left), corresponding GFP-positive frequency (right).Idebenone was used as control drug. E, HTTpQ103-GFP subcellular localization of the inclusions after 18 h induction in SC Galmedium was assessed by fluorescence microscopy with and without R. idaeus (var Prestige) and R. idaeus (var Octavia) extractincubation. Idebenone was used as control drug. F, Clearance of HTTpQ103-GFP evaluated by western blotting at the indicatedtime points with and without R. idaeus (var Prestige) and R. idaeus (var Octavia) extract incubation. Idebenone was used ascontrol drug. Phosphoglycerate kinasewas used for normalization. G, Superoxidemedian fluorescence intensity assessed by flowcytometry after 18-h induction in SC Gal medium with and without R. idaeus (var Prestige) and R. idaeus (var Octavia) extractincubation. For all graphs, data represent themean6 SE of at least three biological replicates. Statistical differences are denoted as*P, 0.05, **P, 0.01, and ***P, 0.001, relative to that in the HTTpQ103-GFP condition. PGK, Phosphoglycerate kinase; SSC,side scatter; ns, not statistically significant; Vis, visual spectrum; MFI, median fluorescence intensity; a.u., arbitrary units; GAE,gallic acid equivalent.


Kallscheuer et al.


http://www.plantphysiol.org/cgi/content/full/pp.18.01074/DC1







and seasonal weather extremes, other means for pro-ducing this compound need to be explored if thesefindings are to be translated to a commercial environ-ment. In that regard, establishing a synthetic biosyn-thetic pathway toward salidroside in microorganismsand a process for its production appears to be a prom-ising strategy for facile access to salidroside.

Pathway Reconstruction for Microbial Production ofSalidroside in S. cerevisiae

In plants of the Rhodiola genus, salidroside is syn-thesized from the aromatic amino acid L-Tyr in threeenzymatic steps (Torrens-Spence et al., 2018). The in-volved glycosyltransferase, catalyzing the last step,uses uridine-diphosphate activated Glc (uridine di-phosphate [UDP]-Glc) as donor for the sugar moiety. S.cerevisiae has an Ehrlich pathway as dedicated routefor degrading aromatic amino acids, which is capableto catabolize L-Tyr directly to tyrosol, the aglyconeof salidroside (Hazelwood et al., 2008; Fig. 6). Thisprompted us to choose this yeast for testing salidrosideproduction by glycosylation of tyrosol obtained fromthe degradation of L-Tyr by using plant-derived UDP-Glc–dependent glycosyltransferases (UGT). In general,metabolic routes for biosynthesis and degradation ofaromatic amino acids, including the Ehrlich pathway,are highly regulated in yeasts. Hence, as expected, apreliminary analysis of the basic S. cerevisiae S288Cyeast strain BG (Eichenberger et al., 2017) showed onlya very low accumulation of 0.02 mM (3 mg L21) tyrosolin the total broth. To deregulate the biosynthesis of theprecursor L-Tyr, we took advantage of feedback-resistant variants of L-Tyr-sensitive 3-deoxy-D-arabi-noheptulosonate 7-P synthase (Aro4) and chorismatemutase (Aro7) of the aromatic amino acid-formingshikimate pathway (Luttik et al., 2008). The two re-spective genes ARO4K229L and ARO7T266I, encoding forthese two enzymes, were integrated into the genome ofthe BG strain, replacing the ARO3 gene encoding a

3-deoxy-D-arabinoheptulosonate 7-P synthase isoen-zyme feedback-regulated by L-Phe. This approach cir-cumvents the natural feedback inhibition by L-Tyr, astrategy that was previously used in our labs to increasethe carbon flow toward L-Tyr (Lehka et al., 2017). Re-lieving the feedback-inhibition resulted in a 10-foldincrease in tyrosol concentration compared to that inthe BG strain. To further improve the supply of tyrosol,we overexpressed two native genes, TYR1 encodingprephenate dehydrogenase, and ARO10 encoding a2-oxo acid decarboxylase. These two genes were inte-grated into the yeast genome under the control ofstrong promoters, resulting in a strain, namely BG4,that producedmore than 3.2mM (442mg L21) of tyrosolin 72 h (Fig. 7). This strain was used for testing UGTs forsalidroside production. For this purpose, two syntheticgenes encoding the glycosyltransferases UGT72B14and UGT73B6 from Rhodiola sachalinensis, which werepreviously applied to glycosylate tyrosol (Ma et al.,2007; Yu et al., 2011), were codon-optimized for ex-pression in yeast and integrated into the strain setup. Inaddition, we performed a rapid in vitro screening ofour in-house collection of purified UGTs for identify-ing variants with the ability to glycosylate tyrosol. Asingle UGT, OsUGT13 from Oryza sativa, showed astrong tyrosol glycosylation activity and the correspond-ing gene was therefore also synthesized as codon-optimized version for a heterologous gene expressionin yeast. The three individual genes were cloned intoyeast expression cassettes and introduced into thetyrosol producing strain BG4 by in vivo assembly ofHRT plasmids as described earlier (Eichenberger et al.,2017). Surprisingly, application of the newly identifiedOsUGT13 resulted in much higher salidroside titerscompared to those of the yeast variants expressing thetwo R. sachalinensis UGTs, as these two strains accu-mulated only very low amounts of salidroside (Fig. 7).In contrast, on a molar ratio, the yeast strain employingOsUGT13 converted more than two-thirds of the pro-duced tyrosol to 2.1 mM (640 mg L21) salidroside in72 h. Notably, salidroside was the only product with no

Figure 3. Fractionation of the R. idaeus(var Prestige) extract. The chromatogramwas obtained by quantification of totalphenols (blue line, left y axis) for thefractions of R. idaeus (var Prestige) ex-tracts. In total, 11 fractions were obtainedby pooling the fractions comprising peaksof total phenols as indicated by the boxes.The boxes indicated in green correspondto fractions promoting the improvement ofcellular growth in the yeastHDmodel as itwas done for the (poly)phenol-enrichedextract. GAE, gallic acid equivalent.





Tab

le1.Annotatedco

mpoundsfoundin

theHD

bioactive

frac

tions5an

d7ofR.idae

us(var

Prestige)

inpositive

andneg

ativemodes

usingaThermoOrbitrapXLLC

-MS.

Additionally,thepresence

(+)orab

sence

(2)ofco

mpoundsin

thefrac

tionsisnoted.

Reten

tion

Tim

e(m

in)

m/z

Molecu

larFo

rmula

Ten

tative

Iden

tification

Iden

tity

Leve

lFrac

tions

Positive

Mode

Neg

ativeMode

57

8.93

—343.1029[M

-H]2

C15H

20O

9Methoxyben

zoyl-hex

oside

3+

—9.17

—299.0769[M

-H]2

C13H

16O

8Hyd

roxy

-ben

zoyl-hex

osideisomer

A3

+—

375.0928

—Unkn

own

4+

—9.31

332.1354[M

+NH

4]+

313.0923[M

-H]2

C14H

18O

8Hyd

roxy

phen

ylac

etate-hexoside

3+

—359.0976[M

+FA

-H]2

10.10

205.0977[M

+H]+

203.0825[M

-H]2

C11H

12N

2O

2Trp

1+

—10.45

—357.1186[M

+FA

-H]2

C15H

20O

7Phen

ylpropan

oyl-hex

oside

3+

—143.0350

—Unkn

own

4+

—10.55

—299.1132[M

-H]2

C14H

18O

8Sa

lidroside

1+

—345.1184[M

+FA

-H]2

10.72

—341.0874[M

-H]2

C15H

18O

9Caffeoyl-gluco

side

2+

—11.03

—427.1239[M

-H]2

C19H

24O

11

Unkn

own

4+

—11.08

—299.0768[M

-H]2

C13H

16O

8Hyd

roxy

-ben

zoyl-hex

osideisomer

B3

+—

355.0666[M

-H]2

C15H

16O

10

Coumaroyl-quinic

acid

2+

—11.41

464.1780[M

+NH

4]+

445.1342[M

-H]2

C19H

26O

12

Ben

zoyl-dihex

osideisomer

A3

+—

469.1333[M

+Na]

+

11.65

—445.1346[M

-H]2

C19H

26O

12

Ben

zoyl-dihex

osideisomer

B3

+—

11.73

611.1619M

+609.1446[M

+-2H]2

C27H

31O

16+

Cyanidin

3-O

-sophoroside

1—

+12.00

757.2214M

+755.2022[M

+-2H]2

C33H

41O

20+

Cyanidin

di-hexosiderham

nosyl

2—

+12.19

449.1098M

+447.0928[M

+-2H]2

C15H

11O

6+

Cyanidin

3-O

-gluco

side

1—

+12.37

595.1672M

+593.1499[M

+-2H

2C27H

31O

15+

Cyanidin

3-O

-rutinoside

1—

+12.40

629.174[M

+H]+

627.1552[M

-H]2

C15H

12O

7Taxifolindi-hex

oside

3—

+12.62

—439.1815

—Unkn

own

4—

+523.1812

—Unkn

own

4—

+623.2181

—Unkn

own

4—

+12.66

808.2535[M

+NH

4]+

789.2075[M

-H]2

C33H

42O

22

Unkn

own

4—

+12.67

231.114

——

Unkn

own

4+

—302.1249

——

Unkn

own

4+

——

329.0873

—Unkn

own

4+

——

577.1344

—Unkn

own

4+

——

283.0821

—Unkn

own

4+

—12.72

467.1197[M

+H]+

465.1031[M

-H]2

C21H

22O

12

Taxifolin-hex

oside

3—

+12.78

544.2408[M

+NH

4]+

—C25H

34O

12

Unkn

own

4—

+12.99

317.1391[M

+H]+

—C18H

20O

5Unkn

own

4—

+13.10

—385.1135[M

-H]2

C17H

22O

10

Sinap

oyl-hex

oside

3—

+—

475.1451[M

-H]2

C20H

28O

13

Hyd

roxy

phen

ylac

etate-dihexoside

4—

+13.15

434.2035[M

+NH

4]+

461.1656[M

+FA

-H]2

C19H

28O

10

Ben

zylalco

hol-rutinoside

3—

+13.22

464.1781[M

+NH

4]+

445.1348[M

-H]2

C19H

26O

12

Ben

zoyl

di-hex

osideisomer

C3

+—

491.1401[M

+FA

-H]2

267.067

——

Unkn

own

4+

—13.40

291.0873[M

+H]+

289.0712[M

-H]2

C15H

14O

6(2

)-ep

icatechin

1—

+13.56

—571.2025[M

-H]2

C26H

36O

14

Unkn

own

4—

+(Tab

leco

ntinues

onfollowingpag

e.)


Kallscheuer et al.



masses detected corresponding to possible side pro-ducts such as the icariside D2 (tyrosol glycosylatedat the phenolic hydroxyl group) or the double-glycosylated tyrosol derivative.

Metabolic Engineering toward Salidroside Productionin C. glutamicum

We decided to also establish the biosynthetic route tosalidroside in C. glutamicum as this organism, alreadyused for the million ton-scale production of the aminoacids such as L-Glu and L-Lys, is characterized by a hightolerance toward toxic aromatic compounds (Kitade et al.,2018; Kogure and Inui, 2018). Not surprisingly, several C.glutamicum strains for the synthesis of aromatic plantnatural products (Kallscheuer et al., 2016, 2017; Bragaet al., 2018) and aromatic amino acids as their precursormolecules are available (Ikeda, 2006), rendering this pro-karyotic platform organism a suitable alternative to yeastfor salidroside production. Initially, the focus was put onthe functional introduction of the glycosyltransferasesUGT72B14 or UGT73B6 from R. sachalinensis for con-verting tyrosol to salidroside. As the metabolism of C.glutamicum is naturally incapable of providing tyrosol,salidroside synthesis was first tested by supplementingtyrosol. Unfortunately, no salidroside production couldbe observed in constructed strains expressing codon-optimized genes for the R. sachalinensis UGTs. Mostlikely, improper folding of the plant enzymes in C. glu-tamicummight abolish glycosyltransferase activity, whichwas also observed earlier during the expression of plant-derived genes (Kallscheuer et al., 2017). Previously,translational fusion of the protein of interest to themaltose-binding protein MalE from E. coli improved theT

able

1.(Continued

from

previouspage.)

Reten

tion

Tim

e(m

in)

m/z

Molecu

larFo

rmula

Ten

tative

Iden

tification

Iden

tity

Leve

lFrac

tions

Positive

Mode

Neg

ativeMode

57

13.70

—299.0770[M

-H]2

C13H

16O

8Unkn

own

4—

+401.1444

—Unkn

own

4—

+445.1347[M

-H]2

C19H

26O

12

Ben

zoyl

di-hex

osideisomer

D3

—+

491.1400[M

+FA

-H]2

14.11

—783.0662[M

-2H]22

C68H

48O

44

Sangu

iinH-10;

2—

+1401.6058[M

-2H]22

C123H

80O

78

Lambertian

inC

2—

+14.16

—371.0975[M

-H]2

C16H

20O

10

Unkn

own

4—

+14.40

—934.0682[M

-2H]22

C82H

54O

52

Sangu

iinH-6

2—

+14.80

—455.1761[M

+FA

-H]2

C17H

30O

11

Unkn

own

4—

+14.82

—859.071[M

-2H]22

C75H

52O

48

Unkn

ownTannin

3—

+

Figure 4. Salidroside areas detected during LC-MS/MS analysis in ex-tracts of various Rubus species. Areas were obtained by integrating theselected ion chromatograms of m/z 345 at the retention time of12.536 min of each extract analyzed in ESI negative mode. Data rep-resent the mean 6 SE of at three technological replicates.





solubility of the plant protein yielding functional enzymesin C. glutamicum. By following the same strategy, accu-mulation of low salidroside concentrations of up to0.02 mM (6 mg L21) from 20 mM supplemented tyrosolcould be achieved with a C. glutamicum strain synthesiz-ing the MalE-UGT73B6 fusion protein, whereas no sali-droside formation could be detected in strain employingthe MalE-UGT72B14 glycosyltransferase.At this stage, it was speculated that low intracellular

availability of the glycosyl-donor UDP-Glc probablylimits tyrosol conversion by C. glutamicum, a problemencountered previously during disaccharide produc-tionwith this microorganism (Padilla et al., 2004). Thus,the strain was additionally engineered toward in-creased UDP-Glc supply. UDP-Glc is obtained fromGlc-1-P (G1P), an isomer of the glycolysis intermediate

Glc-6-P (G6P) provided by phosphoglucomutase(Fig. 6). Subsequently, the uridine triphosphate (UTP):G1P uridylyltransferase (GalU) catalyzes the transfer ofthe uridinemonophosphate (UMP)moiety fromUTP toG1P yielding UDP-Glc (Weissborn et al., 1994). A C.glutamicum strain harboring MalE-UGT73B6 and ad-ditionally expressing the genes pgm and galU from E.coli accumulated 0.12 mM (37 mg L21) salidrosidefrom 20 mM tyrosol pointing toward a bottleneck atthe stage of UDP-Glc supply. The overall yield of0.01 mol salidroside per mol tyrosol was still verylow, indicating that UGT73B6 might be not the bestsuited UGT for an application during salidrosideproduction in C. glutamicum.For an improved biotransformation of tyrosol, we

then also tested the identified OsUGT13 from O. sativa,

Figure 5. Salidroside is the bioactivecompound of R. idaeus (var Prestige)against HD. S. cerevisiae W303-1Acells either expressing HTTpQ103-GFPor harboring the empty vector (control)were pregrown in SC raffinose me-dium. A, Growth curves of cells incu-bated in SC Gal medium for 24 h (left).The confidence interval for the maxi-mum growth rate (m) and final biomassis shown on the right. B, Cell growthassessed after 6 h induction in SC me-dium with Gal by phenotypic growthassays in SC medium with Glc, Gal, orglycerol. C, Superoxide production af-ter 18 h induction in SC medium withGal was determined by flow cytometry.D, Growth of the S. cerevisiae BY4741wild type and sod1mutant strains in thepresence of H2O2 and/or salidroside inSC Glc medium for 24 h. Representa-tive images are shown and the values inall graphs represent the mean6 SE of atleast three biological replicates. Statis-tical differences are denoted as *P ,0.05, **P , 0.01, and ***P , 0.001,relative to that in the HTTpQ103-GFPcondition (C) or in the untreated con-dition (D). MFI, median fluorescenceintensity; a.u., arbitrary units.


Kallscheuer et al.



which allowed for the accumulation of the highest sal-idroside concentrations in cultivations of the engi-neered yeast strains. Functional introduction of theengineered MalE-OsUGT13 fusion protein into the C.glutamicum strain engineered for improved UDP-Glcsupply allowed for a 60-fold increase in salidroside ac-cumulation compared to that in the strain harboringMalE-UGT73B6. Under optimized expression conditions,

3.6 mM (1,100 g L21) salidroside was produced from20 mM tyrosol corresponding to a yield of 0.18 mol sali-droside per mol tyrosol. The highest conversion yield of0.74 mol salidroside per mol tyrosol with a final salidro-side titer of 30 mM (8,900 g L21) was observed with aninitial tyrosol concentration of 40 mM (Supplemental Fig.S4). Tyrosol concentrations exceeding 40 mM had a neg-ative effect on growth, which also negatively affectedtyrosol conversion yields (Supplemental Fig. S4).

At this stage, salidroside production was still de-pendent on the supplementation of tyrosol as precursormolecule. For rendering the production economicallymore viable, we also tested production starting fromL-Tyr. This compound is not only less expensive thantyrosol, but can also be naturally supplied by the hoststrain when using the mentioned strategies for over-production of aromatic amino acids. For enabling sali-droside production from L-Tyr, the Ehrlich pathway forsupplying tyrosol was functionally integrated into C.glutamicum and combined with MalE-OsUGT13. Thissynthetic Ehrlich pathway is composed of the 2-oxoacid decarboxylase Aro10 from S. cerevisiae and the al-cohol dehydrogenase YqhD from E. coli (Vuralhanet al., 2005; Jarboe, 2011; Fig. 6). For the initial trans-amination reaction, we relied on the native expressionof the endogenous transaminases AroT and IlvE in C.glutamicum, which are both known to transaminateL-Tyr (Marienhagen et al., 2005). The heterologouspathway was found to be functional as C. glutamicumexpressing aro10 and yqhD along with the gene encod-ing MalE-OsUGT13 were capable of producing 0.5 mM

(150 mg L21) salidroside when 20 mM L-Tyr was sup-plemented, whereas no salidrosidewas producedwhen

Figure 6. Biosynthetic pathway exploited for salidroside production in S. cerevisiae and C. glutamicum. The direct salidrosideprecursor tyrosol can be obtained from the aromatic amino acid L-Tyr by an Ehrlich pathway (shown in green), which is naturallypresent in S. cerevisiae andwhich was functionally introduced intoC. glutamicum by expression of the heterologous genes aro10and yqhD encoding 2-oxo acid decarboxylase and alcohol dehydrogenase, respectively. The conversion of L-Tyr to 4-hydroxyphenylpyruvate was catalyzed by endogenous transaminases in both organisms. C. glutamicum was also engineeredtoward an increased supply of UDP-Glc required for the glycosylation of tyrosol catalyzed by UDP-Glc-dependent glycosyl-transferases (shown in blue). To this end, genes coding for phosphoglucomutase and GalU were expressed for increasing theconversion of the glycolysis intermediate Glc-6-P to UDP-Glc. Pgm, phosphoglucomutase; TA, transaminase; ODC, 2-oxo aciddecarboxylase; ADH, alcohol dehydrogenase.

Figure 7. Salidroside production in engineered S. cerevisiae. S. cer-evisiae BG4 strains expressing codon-optimized genes encoding thethree indicated uridine-diphosphate–dependent glycosyltransferaseswere cultivated as described in the “Material andMethods” section. Thefinal concentrations of the aromatic amino acids L-Ph and L-Tyr and oftyrosol and salidroside in the culture supernatant obtained after 72 h ofcultivation are shown. Data represent the mean 6 SE of at three bio-logical replicates.








the strain did not harbor aro10 and yqhD. Although asalidroside production strategy from L-Tyr was estab-lished, we decided to use the biotransformation strainwith increased UDP-Glc supply for the up-scaling ex-periments to achieve product quantities sufficient forretesting salidroside in the yeast HD model.

Scale-up, Downstream Processing, and Retestingof Bioactivity

Both organisms, S. cerevisiae andC. glutamicum, couldbe successfully engineered toward producing salidro-side. However, it remained to be demonstratedwhether themicrobially produced salidroside provokesthe same beneficial effects in theHDmodel experimentsas the commercially available salidroside analyticalstandard (purity . 98%). For that purpose, the micro-bial salidroside production was scaled up. In the case ofC. glutamicum, biotransformations in two batch fer-mentations with initial concentrations of 40 g L21 and80 g L21 Glc were performed, each in the presence of40 mM tyrosol. The maximum biomass concentration of24 g L21 obtainedwith 80 g L21 Glc was almost twice ashigh as the biomass concentration of 13 g L21 obtainedfrom 40 g L21 Glc. The highest salidroside titer of 32mM

(9.7 g L21) was achievedwith 80 g L21 of Glc, and underthese conditions the salidroside yield was 0.81 molsalidroside per mol of tyrosol, which is 1.8-fold higherthan the product yield of 0.43 obtained with 40 g L21 ofGlc. The best yield is only 10% higher than the yield of0.74 obtained during shaking flask cultivations, indi-cating that at least for the biotransformation of tyrosolthere is no large impact of the controlled cultivationconditions in the bioreactor. The cultivation of S. cer-evisiae was scaled from the deep-well plate scale to theshaking flask scale for achieving a culture volume suf-ficient for salidroside purification.Salidroside was separately purified from the fer-

mentation broth of S. cerevisiae and C. glutamicum(Supplemental Fig. S5). In direct comparison to thecommercially available salidroside, the microbiallyproduced and purified compoundwas tested in the HDyeast model to confirm that the microbially producedsalidroside has the same beneficial bioactivity. Withrespect to the salidroside-mediated cell protection byreducing superoxide anions, the data indeed showedthat salidroside produced with S. cerevisiae or C. gluta-micum has very similar protective activities comparedto that of the commercial, chemically synthesizedcompound (Fig. 8).

DISCUSSION

Plant natural products represent a rich source of bi-oactive compounds with numerous applications. Thisis reflected in the number of pharmaceuticals, nutra-ceuticals, and food additives brought to market, whichare, to a large extent, natural plant products or

derivatives thereof, or are mimics based on the struc-ture (Marienhagen and Bott, 2013; Kallscheuer et al.,2019). Such compounds are also recognized as prom-ising therapeutics for slowing down the course of NDs.Among these, HD is an inherited disorder resulting inthe death of brain cells, which is associated with prob-lems in coordination and mental abilities (Dayalu andAlbin, 2015). HD is caused by alterations in the geneencoding HTT. This protein harbors a poly-Gln (poly-Q) sequence close to the N terminus, which consists of10 to 35 Gln residues in the normal population but isexpanded to 36 to 120 repeats in HD patients(MacDonald and TheHuntington’s Disease CollaborativeResearch Group, 1993; DiFiglia et al., 1997). The severityof the disease directly increases with the increasing lengthof the poly-Q sequence, and pathological symptoms ap-pear when the threshold of 37 Gln-encoding CAG co-dons is overcome. As a result, proteotoxic aggregates ofpoly-Q-expanded HTT, form in neuronal and glial cells.In our study, we showed that salidroside is an abundantbioactive compound inR. idaeus (var Prestige),whereas itis present in much lower amounts in 14 other Rubusspecies. Bioactivity of the R. idaeus (var Prestige) extractagainst HD could be traced back to salidroside as con-firmed in the analogous bioactivity assays using sali-droside, which was commercially available or producedin engineered S. cerevisiae and C. glutamicum strains.Salidroside was also identified in the Chinese me-

dicinal herb R. sachalinensis (Lu et al., 1980), whichrepresents a major source of this compound (Xu et al.,1998; Wu et al., 2003; Zhou et al., 2007). It was shown to

Figure 8. Salidroside produced in S. cerevisiae and C. glutamicummediated a similar protection as commercially available salidroside.W303-1A cells expressing the gene encoding HTTpQ103-GFP werepregrown in SC raffinose medium and superoxide production after 18 hinduction in SC medium with Gal was determined by flow cytometryusing a DHE probe. Data represent the mean 6 SE of at least three bi-ological replicates. Statistical differences are denoted as **P, 0.01 and***P , 0.001, relative to that in the HTTpQ103-GFP condition. a.u.,arbitrary units. MFI, median fluorescence intensity; a.u., arbitrary units.


Kallscheuer et al.




be a potent antioxidant on hydrogen-peroxide–inducedapoptosis and beta-amyloid–induced oxidative stressin SH-SY5Y cells (Zhang et al., 2007, 2010). Moreover,salidroside shows neuroprotective effects against Gluexcitotoxicity in a primary culture of rat hippocampalneurons and protects cells from hypoglycemia and se-rum limitation in the PC12 cell model (Yu et al., 2008).In Caenorhabditis elegans expressing poly-Q proteins,salidroside exerts its neuroprotective function againstpoly-Q-mediated toxicity via oxidative stress pathways(Xiao et al., 2014). However, the protective effect ob-served for C. elegans oxidative damage was general andnot specific due to poly-Q-mediated toxicity. In thisstudy, we could show that the protective action ofsalidroside is specific against HTTpQ103-GFP-in-duced cell damage and not generally against superoxide-driven damages.

The large potential of salidroside for pharmaceuticalapplications motivated us to design and introducesynthetic routes for its production into industriallyrelevant microorganisms. Yu and coworkers identifiedthe genes coding for glycosyltransferases UGT72B14andUGT73B6 and reported the ability of these enzymesto glycosylate tyrosol yielding salidroside (Yu et al.,2011). Interestingly, the natural pathway in R. sachali-nensis supplying tyrosol remained unidentified for along time. There were indications that tyrosol is pro-duced from L-Tyr (Lan et al., 2013;, however, the com-plete metabolic route to tyrosol in Rhodiola sp. waselucidated only very recently (Torrens-Spence et al.,2018).

With enzymes from yeast and bacteria, a tyrosol bi-osynthetic pathway was reconstructed in E. coli, whichwas subsequently exploited for salidroside productionby additional functional introduction of UGT73B6 intothis strain (Bai et al., 2014). After 48 h, a product titer of0.19 mM (57 mg L21) salidroside could be achieved. Inaddition, 0.21 mM (63 mg L21) of icariside D2, i.e.tyrosol glycosylated at the phenolic hydroxy group,was identified as a major side product. These resultsindicate a rather unspecific glycosylation of tyrosol byUGT73B6. With the glycosyltransferase OsUGT13 fromrice we here identified an alternative UGT, which wasmore suitable for glycosylation of tyrosol both in S.cerevisiae and C. glutamicum. Recently, and after we hadfinished our screening of UGTs, another salidrosidepathway was reconstituted in E. coli, using a combina-tion of various plant genes (Chung et al., 2017). In thisstudy, UGT85A1 from Arabidopsis (Arabidopsis thali-ana), which showed essentially full conversion of tyro-sol to salidroside, allowed for product titers of up to0.97 mM (290 mg L21) after 48 h of cultivation. BecauseArabidopsis is not known to produce salidroside, theseresults demonstrate a relaxed specificity of some UGTsas it was also observed for the enzyme from O. sativaidentified during screening of the UGT library. The sameArabidopsis enzyme (UGT85A1) was recently alsofunctionally introduced into a tyrosol-accumulating S.cerevisiae strain, which was capable of producing2.4 mM (732 mg L21) salidroside during fed-batch

fermentation (Jiang et al., 2018). The obtained salidro-side titer is very similar to the product concentration of2.1 mM (640 mg L21) obtained in our study.

CONCLUSION

This study shows that an interdisciplinary team ofexperts from the fields of plant biology, industrial bio-technology, and analytical chemistry is able to developa complete pipeline for discovery and microbial pro-duction of novel pharmacologically interesting com-pounds of plant origin within a very short time frame;here it took only 24 months from the identification ofsalidroside in R. idaeus (var Prestige) to lab-scale bio-reactor production of this compound in engineeredmicroorganisms. This approach may serve as a blue-print for future campaigns aiming at taking advantageof the chemical diversity of (plant) natural products.

Of course, salidroside, identified as a plant phenolwith bioactivity against HD in this collaborative pro-ject, must be validated in cellular models with a higherdegree of complexity and in preclinical animal modelsto really assess its pharmaceutical potential. Neverthe-less, the microorganisms engineered in this study willensure the availability of sufficient salidroside for thisundertaking.

MATERIALS AND METHODS

Berry Sampling, Extraction Procedure, and Fractionation

Approximately 250 g of Rubus idaeus (var Prestige and Octavia) fresh ripefruits were harvested manually in the field on June 21, 2014 in Invergowrie,Scotland, UK (56.46 latitude, 23.07 longitude). The samples were kept undercool conditions until transfer to 220°C storage. Approximately 150 g of thefrozen fruits were weighed into a solvent-proof blender containing 450 mL ofprecooled 0.2% v/v formic acid in methanol solution. Samples were homoge-nized and subsequently filtered using Whatman filter paper no. 1 (GEHealthcare). A total of ten 1-mL aliquots of the filtrate were solvent-dried usinga miVac duo centrifugal evaporator (Genevac) at room temperature for 4 h,followed by lyophilization, flushed with N2 and stored at 220°C until bioac-tivity assays. In parallel, six 50-mL aliquots of the remaining filtrate were storedat 280°C until fractionation.

The content of the six aliquots of the filtrate was evaporated using a cen-trifugal evaporator (Labconco), solubilized and combined. For the fractionationsan ÄKTA Explorer preparative chromatography system (GE Healthcare) wasused. For each fractionation (a total of eight replicates), 1 mg of gallic acidequivalent was injected into a 31-mL column containing a C18 silica resin(particle size 55–105 mm, pore size 125 Å) utilizing 0.1% formic acid v/v in H2Oas mobile phase A and 100%methanol as mobile phase B. At a flow rate of 3 mLmin21 the following gradient was used: wash step 31 mL; 0%–10% solvent B in10 mL; 10%–40% solvent B in 400 mL; 40%–60% solvent B in 50 mL; a regen-eration step with 100% solvent B for 50 mL. Fractions exceeding 30 mL weresplit. The same fractions from different fractionation replicates were pooled andstored at 280°C. Total phenols of the fractions were determined as describedbelow using the Folin-Ciocalteau method.

LC-MS Analysis of Fractionated Extracts

All obtained fractions were resuspended in 1 mL of 50:50 H2O: acetonitrilewith 0.1% formic acid, subsequently diluted 5-fold with H2O and filtered using0.45-mm filter vials (Thomson Instrument). Subsequently, samples were ana-lyzed by LC-MS using an LC device composed of an Accela 600 quaternarypump and an Accela PDA detector coupled to a linear trap quadropole (LTQ)Orbitrap XL mass spectrometer (Thermo Fisher Scientific) with accurate mass





capabilities, operated under the Xcalibur software package 2.0 as describedpreviously in McDougall et al. (2014). Five microliters of the samples were in-jected into a Synergi Hydro RP-80Å 150 3 2 mm (Phenomenex) and auto-sampler and column temperatures were maintained at 4°C and 30°C,respectively. Samples were eluted at a flow rate of 0.3 mL min21 using twomobile phases (A: 0.1% v/v formic acid in H2O; B: 0.1% formic acid in aceto-nitrile) with the following gradient: 0 min: 2% B; 2 min: 2% B; 5 min: 5% B;25min: 45%B; 26min: 100%B; 29min: 100%B; 30min: 2% B; 35min: 2% B.Massdetection was carried out in both positive and negative ESI modes. Data ac-quisition was performed in ESI full scan mode within the FT detector operatingwith a mass resolution of 30.000 (full width at half-maximum defined at m/z400); the HESI probe temperature was set to 100°C, the capillary temperature at275°C with sheath gas at 60 psi, and auxiliary gas at 30 psi. For the positivepolarity, the source voltage was set at +4.5 kV, capillary voltage at 44 V, andtube lens voltage at 100 V. For negative polarity, the source voltage was set at23.5 kV, capillary voltage at 244 V, and tube lens voltage at 2100 V. In ad-dition, the analysis was repeated in full-scan mode within the FT detector andfollowed by a data-dependent MS/MS of the three most intense ions within theLTQ ion trap detector using Helium as a collision gas, with a normalized col-lision energy of 45 mass isolation width of 6 1 m/z Activation Q of 0.25 andactivation time of 30 ms, source voltage (set at 3.4 kV) in wide band activationmode. Full scan FT data are acquired in profile mode, whereas the DDA MS2LTQ fragmentation spectra are acquired in centroid mode. A scan speed of 0.1 sand 0.4 s are applied in the LTQ and the Fourier transform mass spectrometer,respectively. The Automatic Gain Control was set to 13 105 and 53 105 for theLTQ and the Fourier transform mass spectrometer, respectively. Peaks presentin the bioactive fractions were tentatively annotated by utilizing the accuratemass (,3 ppm), MS/MS fragmentation patterns, and reference to retentiontime for previously analyzed standards when available (SupplementalTable S1).

Total Quantification of Phenolic Compounds

The filtrates of Rubus extracts to be used in the cell assays were solvent-driedusing a Speed-vac, resuspended in CH3COOH/H2O and subsequently sub-jected to solid phase extraction (Tavares et al., 2010). Total phenolic content ofthe samples were determined using the Folin-Ciocalteau method adapted tomicroplate readers (Tavares et al., 2010) with gallic acid as standard. Totalphenolic content was expressed as mg GAEmL21. The samples were aliquoted,freeze-dried, and stored at 220°C.

Yeast Plasmids, Strain, and Transformation forBioactivity Screening

Plasmids and strains used in this study are listed in Supplemental Table S2.For construction of p426_GAL1pr-GFP-AB42, the fragment GFP-AB42 wasremoved from p416_GPD-GFP_AB42 using BamHI and SmaI and cloned intothe p426_GAL1. For construction of p426_GAL1pr-FLAG-HTT103Q-GFP, thesequence GAL1pr-FLAG-HTTp103-GFP from p425GAL1_HTT103Q was am-plified by PCR and cloned into the p426 vector using the In-Fusion Cloning Kit(TAKARA Clontech). W303-1A_FUS andW303-1A_T strains were obtained bytransformation of W303-1A strain with plasmids pAG303_GAL1pr-FUS andpAG303_GAL1pr-ccdB, previously linearized with BstZ17I. Yeast transforma-tion procedures were carried out using a lithium acetate method described inGietz et al. (1995). Synthetic complete (SC)medium [0.67% (w/v) yeast nitrogenbase without amino acids (YNB; Difco) and 0.79 g L21 complete supplementmixture (CSM; QBiogene), containing 1% (w/v) raffinose, was used for growthof PD andALS integrative models. Synthetic dropout CSM-URAmedium [0.67%(w/v) YNB and 0.77 g L21 single amino acid dropout CSM-URA (QBiogene)containing 1% (w/v) raffinose was used for growth of PD model. Syntheticdropout SC-LEU medium 0.6% (w/v) YNB and 0.54 g L21 6-amino aciddropout CSM-ADE-HIS-LEU-LYS-TRP-URA (QBiogene) supplemented with standardconcentrations of the required amino acids and containing 1% (w/v) raffinosewas used for growth of the HDmodel. In all conditions, repression or inductionof the expression of the disease-encoding gene was carried out in mediumcontaining Glc (for repression) andGal (for induction) at a final concentration of2% (w/v). A preinoculumwas prepared in raffinosemedium and cultures wereincubated overnight at 30°C under orbital shaking. Cells were diluted in freshmedium and cultures were incubated under the same conditions until the op-tical density at 600 nm (OD600) reached 0.56 0.05 (log phase). Cell suspensionswere further diluted according to the equation ODi 3 Vi = ODf/(2([t/gt]) 3 Vf,with ODi = initial optical density of the culture, Vi = initial volume of culture,

ODf = final optical density of the culture, t = time (usually 16 h), gt = generationtime of the strain, andVf = final volume of culture to ensure synchronicity of thecells within the cultures.OD600 readings were performed in a 96-well microtiterplate using a Power Wave XS plate spectrophotometer (BioTek Instruments).For the phenotypic growth assays, yeast was grown as described toOD600 0.160.01 andwas inoculated to anOD600 0.26 0.02 in medium supplemented or notwith the indicated concentrations of Rubus extracts or the pure compounds.After 6 h, OD600 was adjusted to 0.05 6 0.005, serial dilutions were performedwith a ratio of 1:3, and 5 mL of each dilution was spotted onto solid mediumcontaining Glc or Gal as the sole carbon source. Growth was recorded after 48 hincubation at 30°C. Images were acquired using Chemidoc XRS (Bio-RadLaboratories) and Quantity-one software (Bio-Rad Laboratories). For thegrowth curves, yeast cultures were diluted to an OD600 of 0.126 0.012 in freshmedium supplemented or not with the indicated concentrations of Rubus ex-tracts, fractions, or the pure compounds in a 96-well microtiter plate. After 2 h ofincubation at 30°C, cultures were further diluted to an OD600 of 0.03 6 0.003 inmedium containing Glc or Gal and supplemented or not with the indicatedcompounds. The cultureswere then incubated at 30°Cwith shaking for 24 h andcellular growth was kinetically monitored hourly by measuring OD600. Datawere modeled using nonlinear parametric regressions and growth parameters(final biomass, maximum growth rate, lag time, doubling time, and area undercurve) as well as % of protection (area under curve relative to the control strain),all with 95% confidence intervals, and were estimated from the best fit modelusing RStudio (Version 0.99.902, GNU lesser general public license). Beforeperforming the bioactivity assays, all extracts were tested for toxicity in therespective control strains. The higher, nontoxic concentration of each extractwas then used for the bioactivity assays.

Flow Cytometry Analysis and Fluorescence Microscopy

Synchronized yeast cell cultures at OD600 of 0.2 6 0.02 were exposed to theindicated concentrations of Rubus extracts or the pure compounds for 18 h inmedium containing Glc or Gal. For analyzing superoxide production using aDHE probe, cells were incubated with 20 mg mL21 of DHE for 30 min at 30°Cprotected from light. Flow cytometry analysis was performed using a CyFlowCube 6 (Sysmex Partec), equipped with a blue solid state laser (488 nm), greenfluorescence channel 530/30 nm, and orange red fluorescence channel 610/20nm. Data analysis was performed using FlowJo software (Ver. 6.7, FlowJo). Aminimum of 30,000 events was collected during each experiment. Results wereexpressed asmedian fluorescence intensity of DHE. In addition, yeast cells werevisually inspected for the formation of intracellular inclusions of HTT by flu-orescence microscopy using a DMRA2 fluorescence microscope (Leica) equip-ped with a CoolSNAP HQ CCD camera (1.3 MPx monochrome). Images wereanalyzed using the software ImageJ (University of WI-Madison).

Immunoblotting

Protein extraction fromyeast cells byglassbead lysis andquantificationof thetotal protein content of each samplewasperformedasdescribed inAusubel et al.(1992) and Gomes et al. (2006). Immunoblotting was carried out as described inMacedo et al. (2015). Antibodies against GFP (Antibodies Incorporated) andPGK (Life Technologies) were used.

Reverse Transcription Quantitative PCR

Reverse transcription quantitative PCR (RT-qPCR) analyseswere performedaccording to the MIQE guidelines (Minimum Information for publication ofQuantitative real-time PCR Experiments; Bustin et al., 2009). Total RNA wasextracted using the E.N.Z.A. yeast RNA extraction Kit (OMEGA Bio-Tek). RNA(200–300 ng) was cleaned and reverse-transcribedwith qScript cDNA superMixKit (Quanta Biosciences). RT-qPCR was performed in a LightCycler 480 In-strument (Roche), using LightCycler 480 SYBR Green I Master (Roche) toevaluate expression of the HTTpolyQ103-GFP fusion gene. Relative standardcurves were constructed for each gene, using triplicate serial dilutions of cDNA.The relative expression of these genes was calculated by the relative quantifi-cation method with efficiency correction, using LightCycler 480 (softwareversion 1.5.0.39; Roche). ACT1 and PDA1 were used as reference genes. Theresults were expressed as fold change mRNA levels relative to that in thecontrol (mRNA fold change) of at least three independent biological replicates.


Kallscheuer et al.






Statistical Analysis

The results related to bioactivity (Figs. 1, 2, 5, and 8) reported in this study arethe average of at least three independent biological replicates and are repre-sented as the mean 6 SE. Differences among treatments were assessed by par-ametric Student’s t test or ANOVA with Tukey’s Honest Significant Differencemultiple comparison test (a = 0.05) using SigmaStat 3.10 (Systat Software).

Salidroside Pathway Reconstruction in Microorganisms

Synthetic genes, based on the protein sequences of Rhodiola sachalinensisUGT72B14 (Acc. no. ACD87062) and UGT73B6 (Acc. no. AAS55083), as well asOryza sativa OsUGT13 (XP_015622802), codon-optimized for expression inSaccharomyces cerevisiae or in Corynebacterium glutamicum ATCC13032, weresynthesized by LifeTechnologies (GeneArt). All constructed strains and plas-mids are listed in Supplemental Table S2. Oligonucleotides used for cloning arelisted in Supplemental Table S3. In case of S. cerevisiae, genes were synthesizedhaving aHindIII restriction site and anAAAKozak sequence at the 59 end and aSacII restriction site at the 39 end. Genes were directly cloned into the yeastexpression cassette of pEVE2176 (BC cassette with pGPD1-gene-tCYC1) forHRT plasmid assembly by in vivo homologous recombination as described inEichenberger et al. (2018). For C. glutamicum, either synthetic genes (coding forthe UGTs mentioned above and of Aro10 from S. cerevisiae and YqhD fromEscherichia coli, all codon-optimized for expression in C. glutamicum) or genomicDNA of E. coli MG1655 served as template for gene amplification. For heter-ologous gene expression in C. glutamicum, provided synthetic genes werereamplified by PCR using primers containing unique restriction sites, whichwere subsequently used for cloning. E. coli DH5a or E. coli XL10 Gold cells(Agilent) were used for cloning of plasmids and were cultivated in LB medium(5 g L21 yeast extract, 10 g L21 tryptone, 5 g L21 NaCl) with 100 mg L21 am-picillin, 50 mg L21 kanamycin or 100 mg L21 spectinomycin. All constructedplasmids were finally verified by DNA sequencing at Eurofins MWG Operon.

Microbial Salidroside Production

The yeast strain BG4 used in this study is a derivative of the S288C strainNCYC 3608 (NCYC). This strain was optimized for the production of aromaticamino acid precursors in the shikimate pathway (Vanegas et al., 2018) andwas additionally engineered toward an improved flow through the Ehrlichpathway (Mikkelsen et al., 2012). S. cerevisiae was grown in 96-well plates at30°C, 5 cm shaking diameter, and 300 rpm for 72 h in SC medium containing1.47 g L21 Synthetic Complete (Kaiser) Drop Out: Leu, His, Ura (Formedium),6.7 g L21 Yeast Nitrogen BaseWithout Amino Acids, 20 g L21 Glc, pH set to 5.8with hydrochloric acid, and supplementedwith 76mgL21 uracil for selection ofthe HRT plasmid. For extraction of relevant compounds, 150 mL culture brothwas mixed with 150 mL acidified methanol (containing 1% hydrochloric acid),incubated for 10 min in a 96-well plate at 30°C, 5 cm shaking diameter, and300 rpm and subsequently clarified by centrifugation at 4,000g for 5 min.

C. glutamicum was cultivated aerobically at 30°C in Brain Heart Infusionmedium (for precultures) or in defined CGXII medium with Glc as sole carbonand energy source (Keilhauer et al., 1993) with kanamycin (25 mg L21) andspectinomycin (100 mg L21) when required for plasmid maintenance. For theproduction of salidroside the constructed strain C. glutamicum DelAro4 wasused as a basis, as it is incapable of degrading aromatic compounds(Kallscheuer et al., 2016). Transformations were performed as described pre-viously in Eggeling and Bott (2005). In the bioreactor cultivations, urea andMOPS were omitted because the pH is automatically controlled. Tyrosol wasdissolved in DMSO. Heterologous gene expression was induced using 1 mM

IPTG 2 h after inoculation. For cultivation experiments C. glutamicum wascultivated for 8–10 h in Brain Heart Infusion medium (first preculture) and wassubsequently inoculated into 50 mL CGXII medium in 500-mL baffled Erlen-meyer flasks (second preculture) with an OD600 of 1 and cultivated overnight.The main cultures were inoculated to an OD600 of 1 in CGXII medium forproduction experiments. Bioreactor cultivations were performed in a 2-LDASGIP Parallel Bioreactor System (Eppendorf). The operating volume forfermentations was 1 L, the temperature set-point was maintained at 30°C, andthe pHwas automatically controlled at 7.0 by addition of 3 MNaOH or 3 M HCl.The dissolved oxygenwas kept above 30% of saturation by using stirring-speedfeedback-control ranging from 650 rpm until 1,500 rpm and a constant air-flowrate of 0.5 volume air per volume medium and min. Formation of foam wasinhibited by addition of Antifoam 204 (Sigma-Aldrich). Biomass concentrations

were determined by measuring theOD600. The obtained values were convertedto biomass dry weight by using a previously prepared calibration curve.

Purification of Salidroside from Fermentation Broth

Salidroside was purified from the fermentation broths of S. cerevisiae and C.glutamicum cultivations. In the case of S. cerevisiae, the broth was first filteredthrough a 0.22-mm filter and then purified by reverse-phase chromatographyusing an ÄKTA Explorer (GE Healthcare). For this purpose, a 30-mL steelcolumn, packed with preparative C18 resin (125 Å and 55–105 mm particle size;Waters) was used. Ethanol served as the eluting agent. Twomobile phases wereapplied (mobile phase A: water, mobile phase B: 40% v/v ethanol). Duringevery run, a 2-mL sample was injected and the flow rate was kept constant at3 mL min21. In the first 40 min, 100% A was applied as washing step. After-ward, a gradient from 0% B to 100% B was applied for 60 min. Salidrosideeluted between 69 and 78 min and a fraction of 15 mL (from 73 to 78 min) wascollected in a 50-mL Falcon tube after each purification cycle. Afterward, thecolumnwas regenerated with 100% ethanol and equilibrated with water for thenext step. The fermentation broth of C. glutamicum was centrifuged (for cellremoval) followed by an ultrafiltration step (for removal of high Mr com-pounds) at 3,000g and for 10 min using a Centriprep YM-3 unit (Merck Milli-pore). Two mobile phases were used (mobile phase A: water, mobile phase B:100% [v/v] ethanol). In every run, a 2-mL sample was injected and the flow ratewas kept constant at 3 mL min21. In the first 40 min, 100% A was applied as awashing step. Afterward, a gradient from 0% B to 100% B was applied for150 min. Salidroside eluted between 69 and 80 min and a fraction of 9 mL wascollected in a 15-mL Falcon tube after each chromatographic cycle. Afterward,the column was regenerated with 100% ethanol and equilibrated for the nextstep with water. After purification, the obtained Falcon tubes were dried bycentrifugal vacuum concentration at 40°C in a RapidVap vacuum evaporationsystem with cold trap (Labconco) until complete drying.

HPLC Analysis for Quantification of Salidroside

Salidroside was quantified using a Shimadzu uHPLC system coupled to aSPD-M20A diode array detector. LC separation was carried out with a Kinetex1.7u C18 100 Å pore size column (50 3 2.1 mm; Phenomenex) at 50°C. Forelution, 0.1% (v/v) acetic acid (solvent A) and acetonitrile supplemented with0.1% (v/v) acetic acid (solvent B) were applied as the mobile phases at a flowrate of 0.4 mL min21. A gradient was used, where the amount of solvent B wasincreased stepwise:mins 0 to 6, 5%–30%;mins 6 to 7, 30%–50%;mins 7 to 8, 50 to100%; and mins 8 to 8.5, 100% –5%. Area values were linear up to metaboliteconcentrations of at least 250 mg L21.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers: KX262844.1 (RsUGT72B14, R. sachalinensis);AY547304.1 (RsUGT73B6. R. sachalinensis); NP_418458.1 (malE, E. coli);NP_415214.1 (pgm, E. coli); NP_415752.1 (galU, E. coli); NM_001180688.3 (aro10,S. cerevisiae), and NP_417484.1 (yqhD, E. coli).

Supplemental Data

The following supplementary materials are available.

Supplemental Figure S1. Compounds with highest relative areas detectedin R. idaeus (var Prestige) compared with that in various Rubus species.

Supplemental Figure S2. Comparison among chromatographic, full MSresults, and MS/MS results of salidroside analytical standard and thecompound tentatively identified as salidroside from fraction 5a in ESInegative mode.

Supplemental Figure S3. Comparison among chromatographic, full MSresults, and MS/MS results of salidroside analytical standard and thecompound tentatively identified as salidroside from fraction 5a in ESIpositive mode.

Supplemental Figure S4. Salidroside production from tyrosol with C.glutamicum.











Supplemental Figure S5. Chromatogram obtained during one chromato-graphic cycle for the purification of salidroside from the fermentationbroth of S. cerevisiae or C. glutamicum.

Supplemental Table S1. Retention times and m/z values utilized for gen-erating selected ion chromatograms for the semiquantification of thecompounds reported in the bioactive fraction 5 and 7 of R. idaeus (varPrestige).

Supplemental Table S2. Strains and plasmids used in this study.

Supplemental Table S3. Oligonucleotides used for cloning and for RT-qPCR.

Supplemental Methods. Identification of a phenol against Huntington’sdisease in raspberries and its microbial production.

ACKNOWLEDGMENTS

We express our gratitude to Dr. Rute Neves (Chr. Hansen A/S, Denmark),Prof. Dr. Jochen Förster and Dr. Alexey Dudnik (The NovaNordisk FoundationCenter for Biosustainability, Technical University of Denmark, Denmark), whocoordinated the BacHBerry project. We also thank Prof. Dr. Ian Macreadie(Centre of Excellence for Alzheimer’s Disease Research & Care, School of Exer-cise, Biomedical &Health Sciences, Edith CowanUniversity,WA, Australia) forproviding p416_GPD-GFP_AB42; Prof. Dr. Tiago Outeiro (University MedicalCenter Gottingen, Department of Neurodegeneration and Restorative Re-search, Germany) for providing W303-1A_Syn and W303-1A TU; Prof. Dr.Flaviano Giorgini (Department of Genetics and Genome Biology, Universityof Leicester, UK) for providing p425GAL1_HTT103Q; and Prof. Dr. GregPetsko (Department of Biochemistry and Chemistry, Rosenstiel BasicMedical Sciences Research Center, Brandeis University, U.S.) for providingpYES_GAL1pr-FUS-GFP and pYES_CT.

Received September 6, 2018; accepted October 22, 2018; published November 5,2018.

LITERATURE CITED

Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman J, Smith JA,Struhl K (1992) Short protocols in molecular biology. John Wiley &Sons, New York

Bai Y, Bi H, Zhuang Y, Liu C, Cai T, Liu X, Zhang X, Liu T, Ma Y (2014)Production of salidroside in metabolically engineered Escherichia coli. SciRep 4: 6640

Bensalem J, Dal-Pan A, Gillard E, Calon F, Pallet V (2015) Protective ef-fects of berry polyphenols against age-related cognitive impairment.Nutr Aging (Amst) 3: 89–106

Bharadwaj P, Martins R, Macreadie I (2010) Yeast as a model for studyingAlzheimer’s disease. FEMS Yeast Res 10: 961–969

Braga A, Oliveira J, Silva R, Ferreira P, Rocha I, Kallscheuer N,Marienhagen J, Faria N (2018) Impact of the cultivation strategy onresveratrol production from glucose in engineered Corynebacteriumglutamicum. J Biotechnol 265: 70–75

Browne SE, Bowling AC, MacGarvey U, Baik MJ, Berger SC, Muqit MM,Bird ED, Beal MF (1997) Oxidative damage and metabolic dysfunctionin Huntington’s disease: Selective vulnerability of the basal ganglia. AnnNeurol 41: 646–653

Browne SE, Ferrante RJ, Beal MF (1999) Oxidative stress in Huntington’sdisease. Brain Pathol 9: 147–163

Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M,Mueller R, Nolan T, Pfaffl MW, Shipley GL, et al (2009) The MIQEguidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55: 611–622

Chung D, Kim SY, Ahn J-H (2017) Production of three phenylethanoids,tyrosol, hydroxytyrosol, and salidroside, using plant genes expressingin Escherichia coli. Sci Rep 7: 2578

Dayalu P, Albin RL (2015) Huntington disease: Pathogenesis and treat-ment. Neurol Clin 33: 101–114

DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP,Aronin N (1997) Aggregation of huntingtin in neuronal intranuclearinclusions and dystrophic neurites in brain. Science 277: 1990–19939302293

Dudnik A, Almeida AF, Andrade R, Avila B, Bañados P, Barbay D,Bassard J-E, Benkoulouche M, Bott M, Braga A, Breitel D, Brennan R,et al. (2018) BacHBerry: BACterial Hosts for production of Bioactivephenolics from bERRY fruits. Phytochem Rev 17: 291–326

Eggeling L, Bott M, editors (2005) Handbook of Corynebacterium gluta-micum. CRCPress, Taylor & Francis Group, Boca Raton, Florida

Eichenberger M, Lehka BJ, Folly C, Fischer D, Martens S, Simón E,Naesby M (2017) Metabolic engineering of Saccharomyces cerevisiae forde novo production of dihydrochalcones with known antioxidant, an-tidiabetic, and sweet tasting properties. Metab Eng 39: 80–89

Eichenberger M, Hansson A, Fischer D, Dürr L, Naesby M (2018) Denovo biosynthesis of anthocyanins in Saccharomyces cerevisiae. FEMSYeast Res 18

Flick JS, Johnston M (1990) Two systems of glucose repression of the GAL1promoter in Saccharomyces cerevisiae. Mol Cell Biol 10: 4757–4769

Gao J, Zhou R, You X, Luo F, He H, Chang X, Zhu L, Ding X, Yan T (2016)Salidroside suppresses inflammation in a D-galactose-induced rat modelof Alzheimer’s disease via SIRT1/NF-kB pathway. Metab Brain Dis 31:771–778

Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on thetransformation of intact yeast cells by the LiAc/SS-DNA/PEG proce-dure. Yeast 11: 355–360

Gomes RA, Vicente Miranda H, Silva MS, Graça G, Coelho AV, FerreiraAE, Cordeiro C, Freire AP (2006) Yeast protein glycation in vivo bymethylglyoxal. Molecular modification of glycolytic enzymes and heatshock proteins. FEBS J 273: 5273–5287

Goswami A, Dikshit P, Mishra A, Mulherkar S, Nukina N, Jana NR(2006) Oxidative stress promotes mutant huntingtin aggregation andmutant huntingtin-dependent cell death by mimicking proteasomalmalfunction. Biochem Biophys Res Commun 342: 184–190

Halvorsen BL, Carlsen MH, Phillips KM, Bøhn SK, Holte K, Jacobs DRJr., Blomhoff R (2006) Content of redox-active compounds (i.e. antiox-idants) in foods consumed in the United States. Am J Clin Nutr 84:95–135

Hazelwood LA, Daran J-M, van Maris AJ, Pronk JT, Dickinson JR (2008)The Ehrlich pathway for fusel alcohol production: A century of researchon Saccharomyces cerevisiae metabolism. Appl Environ Microbiol 74:2259–2266

Ikeda M (2006) Towards bacterial strains overproducing L-tryptophan andother aromatics by metabolic engineering. Appl Microbiol Biotechnol 69:615–626

Indo HP, Yen H-C, Nakanishi I, Matsumoto K, Tamura M, Nagano Y,Matsui H, Gusev O, Cornette R, Okuda T, et al (2015) A mitochondrialsuperoxide theory for oxidative stress diseases and aging. J Clin Bio-chem Nutr 56: 1–7

Jarboe LR (2011) YqhD: A broad-substrate range aldehyde reductase withvarious applications in production of biorenewable fuels and chemicals.Appl Microbiol Biotechnol 89: 249–257

Jiang J, Yin H, Wang S, Zhuang Y, Liu S, Liu T, Ma Y (2018) Metabolicengineering of Saccharomyces cerevisiae for high-level production of sal-idroside from glucose. J Agric Food Chem 66: 4431–4438

Jones JA, Vernacchio VR, Collins SM, Shirke AN, Xiu Y, Englaender JA,Cress BF, McCutcheon CC, Linhardt RJ, Gross RA, et al (2017) Com-plete biosynthesis of anthocyanins using E. coli polycultures. MBio 8:e00621–e17

Ju S, Tardiff DF, Han H, Divya K, Zhong Q, Maquat LE, Bosco DA,Hayward LJ, Brown RH Jr., Lindquist S, et al (2011) A yeast model ofFUS/TLS-dependent cytotoxicity. PLoS Biol 9: e1001052

Kallscheuer N, Vogt M, Stenzel A, Gätgens J, Bott M, Marienhagen J(2016) Construction of a Corynebacterium glutamicum platform strainfor the production of stilbenes and (2S)-flavanones. Metab Eng 38:47–55

Kallscheuer N, Vogt M, Bott M, Marienhagen J (2017) Functional ex-pression of plant-derived O-methyltransferase, flavanone 3-hydroxy-lase, and flavonol synthase in Corynebacterium glutamicum forproduction of pterostilbene, kaempferol, and quercetin. J Biotechnol258: 190–196

Kallscheuer N, Classen T, Drepper T, Marienhagen J (2019) Production ofplant metabolites with applications in the food industry using en-gineered microorganisms. Curr Opin Biotechnol 56: 7–17

Keilhauer C, Eggeling L, Sahm H (1993) Isoleucine synthesis in Coryne-bacterium glutamicum: Molecular analysis of the ilvB-ilvN-ilvC operon.J Bacteriol 175: 5595–5603


Kallscheuer et al.








Kitade Y, Hashimoto R, Suda M, Hiraga K, Inui M (2018) Production of 4-hydroxybenzoic acid by an aerobic growth-arrested bioprocess usingmetabolically engineered Corynebacterium glutamicum. Appl EnvironMicrobiol 84: e02587–17

Kogure T, Inui M (2018) Recent advances in metabolic engineering ofCorynebacterium glutamicum for bioproduction of value-added aromaticchemicals and natural products. Appl Microbiol Biotechnol 102:8685–8705

Koopman F, Beekwilder J, Crimi B, van Houwelingen A, Hall RD, BoschD, van Maris AJ, Pronk JT, Daran J-M (2012) De novo production of theflavonoid naringenin in engineered Saccharomyces cerevisiae. Microb CellFact 11: 155

Lan X, Chang K, Zeng L, Liu X, Qiu F, Zheng W, Quan H, Liao Z, Chen M,Huang W, et al (2013) Engineering salidroside biosynthetic pathway inhairy root cultures of Rhodiola crenulata based on metabolic characteri-zation of tyrosine decarboxylase. PLoS One 8: e75459

Lashuel HA, Overk CR, Oueslati A, Masliah E (2013) The many faces ofa-synuclein: From structure and toxicity to therapeutic target. Nat RevNeurosci 14: 38–48

Lehka BJ, Eichenberger M, Bjørn-Yoshimoto WE, Vanegas KG, Buijs N,Jensen NB, Dyekjær JD, Jenssen H, Simon E, Naesby M (2017) Im-proving heterologous production of phenylpropanoids in Saccharomycescerevisiae by tackling an unwanted side reaction of Tsc13, an endogenousdouble-bond reductase. FEMS Yeast Res 17: fox004

Lim CG, Fowler ZL, Hueller T, Schaffer S, Koffas MA (2011) High-yieldresveratrol production in engineered Escherichia coli. Appl Environ Mi-crobiol 77: 3451–3460

Lu X, Zheng J, Wang Z, Zhao L, Pei Y (1980) Study on the native Rhodiolasachalinensis: The extraction, separation and identification of salidroside.Chin Tradit Herbal Drugs 11: 147–148

Luttik MA, Vuralhan Z, Suir E, Braus GH, Pronk JT, Daran JM (2008)Alleviation of feedback inhibition in Saccharomyces cerevisiae aromaticamino acid biosynthesis: Quantification of metabolic impact. Metab Eng10: 141–153

Ma L-Q, Liu B-Y, Gao D-Y, Pang X-B, Lü S-Y, Yu H-S, Wang H, Yan F, LiZ-Q, Li Y-F, et al (2007) Molecular cloning and overexpression of a novelUDP-glucosyltransferase elevating salidroside levels in Rhodiola sacha-linensis. Plant Cell Rep 26: 989–999

MacDonald M; The Huntington’s Disease Collaborative Research Group(1993) A novel gene containing a trinucleotide repeat that is expandedand unstable on Huntington’s disease chromosomes. Cell 72: 971–9838458085

Macedo D, Tavares L, McDougall GJ, Vicente Miranda H, Stewart D,Ferreira RB, Tenreiro S, Outeiro TF, Santos CN (2015) (Poly)phenolsprotect from a-synuclein toxicity by reducing oxidative stress and pro-moting autophagy. Hum Mol Genet 24: 1717–1732

Marienhagen J, Bott M (2013) Metabolic engineering of microorganisms forthe synthesis of plant natural products. J Biotechnol 163: 166–178

Marienhagen J, Kennerknecht N, Sahm H, Eggeling L (2005) Functionalanalysis of all aminotransferase proteins inferred from the genome se-quence of Corynebacterium glutamicum. J Bacteriol 187: 7639–7646

Mason RP, Giorgini F (2011) Modeling Huntington disease in yeast: Per-spectives and future directions. Prion 5: 269–276

Masters CL, Bateman R, Blennow K, Rowe CC, Sperling RA, CummingsJL (2015) Alzheimer’s disease. Nat Rev Dis Primers 1: 15056

McDougall GJ, Conner S, Pereira-Caro G, Gonzalez-Barrio R, Brown EM,Verrall S, Stewart D, Moffet T, Ibars M, Lawther R, et al (2014)Tracking (Poly)phenol components from raspberries in ileal fluid.J Agric Food Chem 62: 7631–7641

Menezes R, Tenreiro S, Macedo D, Santos CN, Outeiro TF (2015) Fromthe baker to the bedside: Yeast models of Parkinson’s disease. MicrobCell 2: 262–279

Mikkelsen MD, Buron LD, Salomonsen B, Olsen CE, Hansen BG,Mortensen UH, Halkier BA (2012) Microbial production of in-dolylglucosinolate through engineering of a multi-gene pathway in aversatile yeast expression platform. Metab Eng 14: 104–111

Milke L, Aschenbrenner J, Marienhagen J, Kallscheuer N (2018) Pro-duction of plant-derived polyphenols in microorganisms: Current stateand perspectives. Appl Microbiol Biotechnol 102: 1575–1585

Niedzielska E, Smaga I, Gawlik M, Moniczewski A, Stankowicz P, Pera J,Filip M (2016) Oxidative stress in neurodegenerative diseases. MolNeurobiol 53: 4094–4125

Nile SH, Park SW (2014) Edible berries: Bioactive components and theireffect on human health. Nutrition 30: 134–144

Oliveira AV, Vilaça R, Santos CN, Costa V, Menezes R (2017) Exploringthe power of yeast to model aging and age-related neurodegenerativedisorders. Biogerontology 18: 3–34

Outeiro TF, Lindquist S (2003) Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science 302: 1772–1775

Padilla L, Morbach S, Krämer R, Agosin E (2004) Impact of heterologousexpression of Escherichia coli UDP-glucose pyrophosphorylase on tre-halose and glycogen synthesis in Corynebacterium glutamicum. ApplEnviron Microbiol 70: 3845–3854

Rodrigues-Pousada C, Nevitt T, Menezes R (2005) The yeast stress re-sponse. Role of the Yap family of b-ZIP transcription factors. ThePABMB Lecture delivered on 30 June 2004 at the 29th FEBS Congress inWarsaw. FEBS J 272: 2639–2647

Rodriguez-Mateos A, Heiss C, Borges G, Crozier A (2014) Berry (poly)phenols and cardiovascular health. J Agric Food Chem 62: 3842–3851

Rosado-Ramos R, Godinho-Pereira J, Figueira I, Jardim C, Garcia G,Menezes R (2018) Exploring the benefits of cellular models to uncoverbioactive polyphenols for neurodegeneration. Curr Pharm Des 24:2076–2106

Seeram NP (2012) Emerging research supporting the positive effects ofberries on human health and disease prevention. J Agric Food Chem 60:5685–5686

Tavares L, Fortalezas S, Carrilho C, McDougall G, Stewart D, Ferreira R,Santos C (2010) Antioxidant and antiproliferative properties of straw-berry tree tissues. J Berry Res 1: 3–12

Torrens-Spence MP, Pluskal T, Li F-S, Carballo V, Weng J-K (2018)Complete pathway elucidation and heterologous reconstitution ofRhodiola salidroside biosynthesis. Mol Plant 11: 205–217

Tsuda T (2016) Recent progress in anti-obesity and anti-diabetes effect ofberries. Antioxidants 5: 13

Vanegas K, Larsen A, Eichenberger M, Fischer D, Mortensen U, NaesbyM (2018) Indirect and direct routes to C-glycosylated flavones in Sac-charomyces cerevisiae. Microb Cell Fact 17: 107

Van Raamsdonk JM, Vega IE, Brundin P (2017) Oxidative stress in neu-rodegenerative disease: Causation or association? Oncotarget 8:10777–10778

Vuralhan Z, Luttik MA, Tai SL, Boer VM, Morais MA, Schipper D,Almering MJ, Kötter P, Dickinson JR, Daran J-M, et al (2005) Physi-ological characterization of the ARO10-dependent, broad-substrate-specificity 2-oxo acid decarboxylase activity of Saccharomyces cerevisiae.Appl Environ Microbiol 71: 3276–3284

Wang S, He H, Chen L, Zhang W, Zhang X, Chen J (2015) Protective effectsof salidroside in the MPTP/MPP+-induced model of Parkinson’s diseasethrough ROS-NO-related mitochondrion pathway. Mol Neurobiol 51:718–728

Weissborn AC, Liu Q, Rumley MK, Kennedy EP (1994) UTP: Alpha-D-glucose-1-phosphate uridylyltransferase of Escherichia coli: Isolation andDNA sequence of the galU gene and purification of the enzyme.J Bacteriol 176: 2611–2618

Willingham S, Outeiro TF, DeVit MJ, Lindquist SL, Muchowski PJ (2003)Yeast genes that enhance the toxicity of a mutant huntingtin fragment ora-synuclein. Science 302: 1769–1772

Wu S, Zu Y, Wu M (2003) High yield production of salidroside in thesuspension culture of Rhodiola sachalinensis. J Biotechnol 106: 33–43

Xiao L, Li H, Zhang J, Yang F, Huang A, Deng J, Liang M, Ma F, Hu M,Huang Z (2014) Salidroside protects Caenorhabditis elegans neurons frompolyglutamine-mediated toxicity by reducing oxidative stress. Mole-cules 19: 7757–7769

Xu J, Liu C, Han A, Feng P, Su Z (1998) Strategies for the improvement ofsalidroside production in cell suspension cultures of Rhodiola sachali-nensis. Plant Cell Rep 17: 288–293

Yang B, Kortesniemi M (2015) Clinical evidence on potential health ben-efits of berries. Curr Opin Food Sci 2: 36–42

Yu H-S, Ma L-Q, Zhang J-X, Shi G-L, Hu Y-H, Wang Y-N (2011) Char-acterization of glycosyltransferases responsible for salidroside biosyn-thesis in Rhodiola sachalinensis. Phytochemistry 72: 862–870

Yu S, Liu M, Gu X, Ding F (2008) Neuroprotective effects of salidroside inthe PC12 cell model exposed to hypoglycemia and serum limitation. CellMol Neurobiol 28: 1067–1078





Zarei S, Carr K, Reiley L, Diaz K, Guerra O, Altamirano PF, Pagani W,Lodin D, Orozco G, Chinea A (2015) A comprehensive review ofamyotrophic lateral sclerosis. Surg Neurol Int 6: 171

Zhang L, Yu H, Sun Y, Lin X, Chen B, Tan C, Cao G, Wang Z (2007)Protective effects of salidroside on hydrogen peroxide-induced apo-ptosis in SH-SY5Y human neuroblastoma cells. Eur J Pharmacol 564:18–25

Zhang L, Yu H, Zhao X, Lin X, Tan C, Cao G, Wang Z (2010) Neuro-protective effects of salidroside against beta-amyloid-induced oxidativestress in SH-SY5Y human neuroblastoma cells. Neurochem Int 57:547–555

Zhou X, Wu Y, Wang X, Liu B, Xu H (2007) Salidroside production by hairyroots of Rhodiola sachalinensis obtained after transformation with Agro-bacterium rhizogenes. Biol Pharm Bull 30: 439–442


Kallscheuer et al.



identification and microbial production of the raspberry ...identiﬁcation and microbial production...

Documents