BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Pathway redesign for deoxyviolacein biosynthesisin Citrobacter freundii and characterization of this pigment

Pei-xia Jiang & Hai-sheng Wang & Su Xiao &

Ming-yue Fang & Rui-ping Zhang & Shu-ying He &

Kai Lou & Xin-Hui Xing

Received: 11 November 2011 /Revised: 8 February 2012 /Accepted: 9 February 2012 /Published online: 6 March 2012# Springer-Verlag 2012

Abstract Violacein (Vio) is an important purple pigmentwith many potential bioactivities. Deoxyviolacein, a struc-tural analog of Vio, is always synthesized in low concentra-tions with Vio in wild-type bacteria. Due to deoxyviolacein’slow production and difficulties in isolation and purification,little has been learned regarding its function and potentialapplications. This study was the first effort in developing astable and efficient biosynthetic system for producing puredeoxyviolacein. A recombinant plasmid with vioabce geneswas constructed by splicing using an overlapping extension-polymerase chain reaction, based on the Vio-synthesizing

gene cluster of vioabcde, originating from Duganella sp. B2,and was introduced into Citrobacter freundii. With the viodgene disrupted in the Vio synthetic pathway, Vio productionwas completely abolished and the recombinant C. freun-dii synthesized only deoxyviolacein. Interestingly, vioegene expression was strongly stimulated in the viod-deleted recombinant strain, indicating that viod disrup-tions could potentially induce polar effects upon thedownstream vioe gene within this small operon. Deox-yviolacein production by this strain reached 1.9 g/L inshaker flasks. The product exhibited significant acid/alkali and UV resistance as well as significant inhibitionof hepatocellular carcinoma cell proliferation at lowconcentrations of 0.1–1 μM. These physical characteristicsand antitumor activities of deoxyviolacein contribute to illu-minating its potential applications.

Keywords Violacein . Deoxyviolacein . Biosynthesis .

Heterologous expression . Pathway redesign

Introduction

Recently characterized as a class of L-tryptophan-derivedalkaloids, bisindoles have attracted much attention due totheir structural features and wide range of biological activ-ities, which suggest potential therapeutic applications (Ryanand Drennan 2009). Despite this great interest, there hasbeen a paucity of studies regarding the isolation of newbisindole molecules from natural sources or the synthesisof derivatives for comparison of their bioactivities and thestudy of bisindoles’ mechanism(s) of action (Ryan et al.2008).

Violacein (Vio), the first reported bisindole compound(Fig. 1), is a purple pigment produced by some bacteria and

Electronic supplementary material The online version of this article(doi:10.1007/s00253-012-3960-0) contains supplementary material,which is available to authorized users.

P.-x. JiangDepartment of Industrial Microbiology and Biotechnology,Institute of Microbiology, Chinese Academy of Sciences,Beijing 100101, People’s Republic of China

P.-x. Jiang : S. Xiao :M.-y. Fang :R.-p. Zhang :X.-H. Xing (*)Institute of Biochemical Engineering,Department of Chemical Engineering, Tsinghua University,Beijing 100084, People’s Republic of Chinae-mail: xhxing@mail.tsinghua.edu.cn

H.-s. WangGraduate School of Chinese Academy of Agricultural Sciences,Beijing 100081, People’s Republic of China

S.-y. HeSchool of Life Science and Technology,China Pharmaceutical University,Nanjing 210009, People’s Republic of China

K. LouInstitute of Microbiology,Xinjiang Academy of Agricultural Sciences,Ürümqi 830091, People’s Republic of China

Appl Microbiol Biotechnol (2012) 94:1521–1532DOI 10.1007/s00253-012-3960-0

displays various important biological activities, including an-tibacterial (Cazoto et al. 2011; Nakamura and Sawada 2003),antifungal (Becker et al. 2009), antiviral (Andrighetti-Frohneret al. 2003), apoptosis-inducing properties in cancer cells(Bromberg et al. 2005; Ferreira et al. 2004), antioxidant(Konzen et al. 2006), trypanocidal (Durán et al. 1994),antidiarrheal (Antonisamy et al. 2009), analgesic andantipyretic (Antonisamy and Ignacimuthu 2010), andantimalarial effects (Duran et al. 2007). Recently, ithas been reported that intraperitoneal doses of Vio upto 1 mg kg−1 are not toxic in mouse blood, kidneys, or liver,resolving the main issues regarding Vio cytotoxicity to severaltumor models in vivo and providing good reference materialfor the use in vivo of Vio and its derivatives as a therapeuticcompound with few side effects (Cazoto et al. 2011).

In the past several years, it has been elucidated that thegene cluster (vioabcde) responsible for Vio biosynthesis iscomposed of five open reading frames and that Vio biosyn-thesis is modulated by a complex metabolic pathway ofVioA→VioB→VioE→VioD→VioC (Fig. 1), carried bythe enzymes of VioABCDE (Balibar and Walsh 2006;Sanchez et al. 2006; Shinoda et al. 2007), which involvesseveral spontaneous (auto-oxidation) reactions (Momen andHoshino 2000). Although the biosynthesis and function of Viohave been studied for decades, there is little understanding ofthe details and regulation mechanism of Vio biosynthesis,which appears to involve several proteins (Balibar and Walsh2006), except that it has been reported that Vio biosynthesisresponds to quorum-sensing signaling molecules (Morohoshiet al. 2010; Shinoda et al. 2007).

In wild Vio-producing bacteria, deoxyviolacein, a Vioderivative, is always produced concurrent with Vio at 3–10-fold lower quantities than Vio (Matz et al. 2008). Due tothe complexity in isolating and purifying deoxyviolaceinfrom crude Vio mixtures, it is difficult to obtain sufficientamounts of pure deoxyviolacein for functional analysis andexploration of its potential application. VioD, a flavin hy-droxylase belonging to the family of flavin-containing NAD(P)H-dependent monooxygenases, controls Vio synthesis(Sanchez et al. 2006). Although it has been inferred thatthe deletion of the putative oxidase viod gene may lead todisappearance of Vio and the production of pure deox-yviolacein, to date, the physiological reason that deoxyviola-cein is always biosynthesized along with Vio production aswell as its exact functional characteristics remain poorlyunderstood.

At present, no information can be obtained regarding theefficient deoxyviolacein production using the viod deletionmethod, no trial has been done for the redesign of deoxy-violacein biosynthetic pathway, and deoxyviolacein has alsonot been widely studied for its bioactivity and functionalcharacteristics, except for its antiprotozoal activity (Matz etal. 2008). It is possible that the Vio biosynthetic intermedi-ate and metabolic pathway of L-tryptophan, the only pre-cursor in Vio biosynthesis, impacts Vio biosynthesis(Antonio and Creczynski-Pasa 2004; Jiang et al. 2010),but this has not yet been reported. VioD enzyme hydroxy-lates protodeoxyviolaceinic acid (PVA), but not the relatedprodeoxyviolacein (PDV), to violaceinic acid (Shinoda et al.2007). The deletion of the viod gene would possibly lead to

Fig. 1 Biosynthetic pathway of L-tryptophan to Vio (dashed area) and deoxyviolacein with viod deleted (Jiang et al. 2010)

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accumulation of the intermediates, such as PDV, PVA, andchromopyrrolic acid, which will then exert a regulatory roleon the Vio pathway. A reliable way to exploit the potentialviod gene regulatory role and the effective production ofdeoxyviolacein is to establish a heterologous productionsystem using convenient host cells capable of expressingexogenous vio genes.

Identification of the multiple catalytic steps in the Viobiosynthetic pathway and the corresponding enzymaticcharacterization offer the necessary information for con-structing a heterologous Vio production system and forobtaining structural analogs of Vio through gene technolo-gy, which further aids in understanding the exact syntheticpathway or improving the amounts of desired metabolites(Balibar and Walsh 2006; Sanchez et al. 2006; Asamizu etal. 2007). Though Escherichia coli with pLvioABCE (viod-deleted Vio gene cluster) completely abolished Vio produc-tion, the appearance of minor amounts of intermediatesPDV accompanying deoxyviolacein will possibly exert aregulatory role on the Vio pathway and lead to inefficientproduction of deoxyviolacein (Sanchez et al. 2006). Recently,the stable and efficient production of crude Vio (at ≥1.6 gL−1)in recombinant Citrobacter freundii has been successfullyachieved by expressing vioabcde from Duganella sp. B2 byour group by using glycerol as carbon source which wasreported to stimulate Vio production (Wang et al. 2009; Jianget al. 2010). C. freundiimay be a suitable host for the efficientsynthesis of violacein and its derivatives own to the presenceof multiple resistance genes and class 1 integrons in C. freun-dii (Ahmed and Shimamoto 2008; Srinivasan et al. 2008),which might play an important role in resisting the antimicro-bial activity of metabolites such as violacein, therebyleading to the high productivity. Moreover, the recom-binant C. freundii can effectively produce the mixture ofviolacein and deoxyviolacein, and the proportion ofdeoxyviolacein in the total pigments produced wasmuch higher than that produced by the original strainDuganella sp. B2, suggesting that production of puredeoxyviolacein could also be feasible by using C. freundii asthe host when viod can be deleted. This heterologous produc-tion system will also be useful to investigate the viod generegulatory role for Vio synthesis.

In this study, based on the vio gene cluster reported in ourprevious paper (Jiang et al. 2010), a recombinant plasmidwith a viod-deleted vio gene cluster, vioabce (vioΔd), wasconstructed by splicing using an overlapping extension-polymerase chain reaction (SOE-PCR) (Kuwayama et al.2002). When the plasmid was introduced into C. freundii,pure deoxyviolacein was produced, as only deoxyviolaceinsynthesis was possible due to the absence of viod in the Viosynthetic pathway, and the yield reached 1.9 gL−1. Physicalcharacteristics and bioactivity assays were then carried outon the pure deoxyviolacein. The pathway redesign of deox-yviolacein biosynthesis permits an expansion of the toolboxthat can be utilized for detailed studies of deoxyviolaceinbioactivities and the regulation mechanism of Vio biosyn-thesis, thus further offering opportunities for comparingbioactivities for Vio and its derivatives and for studyingthe molecular action mechanism(s) of Vio.

Materials and methods

Bacterial strains, culture conditions, and plasmids

The strains and plasmids used in this study are shown inTable 1. C. freundii ACCC 05411 was used as the host forheterologous deoxyviolacein production and recombinantC. freundii (pComvio) was used as the Vio-producing strain(Jiang et al. 2010).

C. freundii ACCC 05411 and C. freundii (pComvio) weregrown in E2 medium (minimal salt medium supplementedwith metal traces: NaH2PO4·2H2O, 0.13%; Na2H-PO4·12H2O, 0.3%; NH4C1, 0.089%; KH2PO4·3H2O,0.75%; 100 mM MgSO4·7H2O, 1%; glycerol, 0.3%, w/v;yeast extract, 0.1%; L-tryptophan, 0.07%; and pH 6.5) in ashaking incubator at 200 rpm (Jiang et al. 2010). Kanamycin(50 μg mL−1) was used in experiments when necessary.

A HepG2 cell line was obtained frozen in liquid nitrogen(−180°C) from the Department of Pharmacology, ChinaPharmaceutical University and cultured in Dulbecco’s Mod-ified Eagle’s Medium (ATCC) containing 10% newborn calfserum (NCS) with 100 UmL−1 penicillin and 100 μg mL−1

streptomycin. The cells were subcultured for 2 days.

Table 1 Strains and plasmidsused in this study Resource Characteristics Source

Strains

C. freundii ACCC 05411 Wild type ACCC

C. freundii (pComvio) Genetic engineering strain Jiang et al. (2010)

E. coli DH10B E. coli cloning strain Stratagene

Plasmids

pCom10 E. coli–Pseudomonas shuttle expression vector Smits et al. (2001)

pComvio pCom10 with vio gene cluster Jiang et al. (2010)

Appl Microbiol Biotechnol (2012) 94:1521–1532 1523

Design and gene assembly for viod gene disruptionin the vio gene cluster

SOE-PCR was used to create genes capable of synthesizingonly deoxyviolacein (Kuwayama et al. 2002), based on theVio gene nucleotide sequence (GenBank accession no.GQ266676) from Duganella sp. B2, as described previously(Jiang et al. 2010). The fragment with a truncated viob geneand full-length vioc and vioe genes were amplified, respec-tively, from Duganella sp. B2 genomic DNA using the HighFidelity PCR system (Takara Biotechnology Co., China)and then assembled by SOE-PCR to produce a 3.1-kb viobcefragment with the primers vio3046-for (binds within viob),vioC-m-rev (binds to the end region downstream of vioc),vioE-m-for (binds to the end region upstream of vioe), andvioE-rev (binds to a region downstream of vioe, SupplementalFig. 1); there were 48 bp overlaps between the primers vioC-m-rev and vioE-m-for. The 3.1-kb SOE-PCR product viobcewas gel-extracted (QIAquick gel extraction kit, Qiagen, Chi-na) and subcloned into pGEM-T Easy (Promega Corp., USA).The total genomic DNA of Duganella sp. B2 was preparedusing Genomic-tip 500/G (Qiagen) and the plasmid DNAfrom both E. coli and C. freundii cells purified using theQiaprep Spin Plasmid Kit (Qiagen). Positive transformationclones were confirmed by sequencing (Invitrogen Corp.,China). All oligonucleotides used for PCR amplification aredescribed in Table 2 and were synthesized by the InvitrogenCustom Primer Facility (Invitrogen Corp.).

Cloning and expression of the vioΔd gene(s) in C. freundii

The primers vioA-for and vioB-4061 were used to amplifythe vioab fragment with a full-length vioa gene and partial-length viob gene. The vioab and viobce fragments weredigested, respectively, with the restriction enzymes AseI/NotI and NotI/XhoI and ligated to produce the 6.1-kb full-length vio genes with viod deleted (vioΔd), which was thencloned into the Pseudomonas–E. coli shuttle vector pCom10at the NdeI and XhoI sites, creating pComvioΔd (Supple-mental Fig. 1b). The vector pCom10 contained the alkane-

responsive promoter PalkB and its positive regulator AlkSfor the regulation of the PalkB promoter (Smits et al. 2001).In a previous experiment, pCom10 was found to be a usefulvector for vio gene expression in C. freundii, and vio genecluster expression facilitated by the Alk promoter is thusregulated by the transcriptional activator AlkS and is in-duced by n-octane (Jiang et al. 2010). pComvioΔd wastransformed into glycerol-competent C. freundii by electro-poration and the transformants were screened using PCRamplification with the vio3046-for and vioE-rev primers,yielding a 3.1-kb fragment that confirmed viod deletion.

Slightly modified from previous fermentation protocols(Jiang et al. 2010), deoxyviolacein was produced by recom-binant C. freundii strain in 20 mL of fermentation mediacontaining 0.05% n-octane (v/v). Broth aliquots of 1.5 mLwere withdrawn after fermentation for 30 h and sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to confirm foreign protein expression inthe engineered cells (pComvioΔd).

Production, extraction, and measurement of deoxyviolaceinproduced by recombinant C. freundii (pComvioΔd)

Cultures were initiated and incubated overnight with 2% (v/v)of an inoculum of uninduced cells. Expression of the vioΔdgene(s) was induced by the addition of n-octane, as above,when a culture’s OD660 reached 1.0. All cultures were per-formed in 100-mL Erlenmeyer flasks containing 20 mL of E2medium on a horizontal shaker at 37°C and the temperatureshifted to 20°C after n-octane was added. After 30 h offermentation, 5 mL aliquots of broth were withdrawn foranalysis.

Deoxyviolacein was extracted from the sampled cells asdescribed previously (Jiang et al. 2010; Wang et al. 2009),but with the following modifications. After fermentation,3 mL of broth was collected and centrifuged at 12,000×gfor 5 min (unless stated otherwise), and the clear supernatantwas discarded. The cell pellets were then rinsed with deion-ized water, centrifuged to recover the cells, resuspended inethanol, and centrifuged again, thus recovering the cells and

Table 2 Synthetic oligonucleotide primers used for PCR amplification

Primer Sequencea Commentsb

vioA-for 5′-GGATCATTAATGACAAATTATTCTGACATTTGCATAG-3′ pComvio¸d

vio3054-rev 5′-AAGAGTGGACTTGGCGGCCGCTTCGACCTG-3′ pComvio¸d

vio3046-for 5′-TACATGACTCAGGTCGAAGCGGCCGCCAAG-3′ pComvio¸d

vioC-m-for 5′-TGGCGTGCGGTGGCATGGCGTCTCCTTAGTTTACCCTTCCAAGTTTGTACC-3′ pComvio¸d

vioE-m-for 5′-GGTACAAACTTGGAAGGGTAAACTAAGGAGACGCCATGCCACCGCACG-3′ pComvio¸d

vioE-rev 5′-GGAATGTCCTCGAGTTCCGACACGAAAACGCTGGC-3′ pComvio¸d

a Restriction enzyme sites underlinedb PCR products used for isolating vio genes and constructing recombinant plasmids for protein expression

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yielding an ethanol extract supernatant containing deoxy-violacein. Ethanol extraction was repeated until the cellswere completely bleached, and the resulting supernatantswere combined. The deoxyviolacein concentration in theextract was determined by measuring the extract’s Abs560(deoxyviolacein extinction coefficient015.955 Lg−1 cm−1

in ethanol at 560 nm), using an ultraviolet–visible spectro-photometer (Beckman DU800, Beckman Instruments, Inc.,USA).

Identification of deoxyviolacein produced by recombinantC. freundii (pComvioΔd)

Extracted crude deoxyviolacein was separated and purifiedusing high performance liquid chromatography (HPLC,Waters 2695, Waters Corp., USA) with an ODS column(Inertsil ODS-35 μm, 250×4.6 mm, Dikma Technologies,Inc., USA) to determine the components (Wang et al. 2009).The conditions were a mobile phase of 70% methanol,1 mL min−1 flow rate, 30°C, and the effluent was monitoredat 560 nm. The resulting eluted fractions were evaporatedunder vacuum at 60°C.

The chemical structure of the extracted deoxyviolacein wasdetermined by HPLC-MS (LCQ Deca XP, Thermo Finnigan,LLC, USA), using electrospray ionization with a spray volt-age of 3.5 kV and mass spectra speed of 0.1 mL min−1. Thedeoxyviolacein identity was further confirmed by 1H-NMRand 13C-NMR (400 MHz, Bruker AV400) after the purifiedpigment was dissolved in dimethyl sulfoxide (DMSO).

Fermentation kinetics of recombinant C. freundii(pComvioΔd)

The transformed C. freundii strain (pComvioΔd) was cultivat-ed as described above, with the inoculum incubated at 37°C at200 rpm for 13–16 h before use to inoculate the fermentationmedium. The LB medium was used to prepare 2% (v/v)inoculums, and after inoculation, n-octane inducer was addedto the fermentation culture when the OD660 reached 1.0, atwhich time the cultivation temperature was shifted to 20°C.Samples were withdrawn during fermentation to measure theconcentrations of glycerol, L-tryptophan, and crude deoxy-violacein (OD560) as well as the cell concentration (OD660).The cell dry weight was calculated from the OD660, based on astandard curve for OD660 and cell dry weight. An HPLCsystem (Waters 2695) with a refractive index detector wasused to determine glycerol concentrations.

Quantitative real-time PCR

For real-time reverse transcription PCR (RT-PCR) experi-ments, 1% (v/v) of precultures of the C. freundii (pCom-vioΔd) and C. freundii (pComvio) strains described above

were inoculated into 100-mL shaker flasks containing20 mL E2 medium (Jiang et al. 2010) and grown to theirlogarithmic phase (12 h) and late exponential phase (32 h)on a rotary shaker. The cells were then harvested by centri-fugation at 10,000×g and 4°C for 1 min for total RNAisolation. Total RNAwas extracted using a RNAiso reagentand then treated with DNase I (Takara Biotech, China). EachRNA sample was quantified using a NanoDrop 2000 spec-trophotometer (Shanghai Fisher Scientific, China) and ad-justed to the same concentration based on absorbance value.For transcriptional analysis of vioe gene, RT-PCR was car-ried out with a first-strand cDNA synthesis kit (Promega,USA), using total RNA as the template. Quantitative geneexpression analysis was performed by quantitative real-timePCR, using the StepOnePlus™ Real-Time PCR System(Applied Biosystems, USA) using the primers 5′-GGAGCAGCGCCTACATCTCGTATT-3′ and 5 ′-CTCCCTGGCATAAGCGACTTTCTG-3′. All measurements werefrom duplicate independent experiments. The 16S rRNAgene was used as an internal standard, obtained using theprimers 5′-GTATGAAGGCGACCGTGAA-3′ and 5′-TATCTGGATGGCGACGAAT-3′.

Acid/alkali and UV resistance of deoxyviolacein

Crude Vio (Vio with 60% of deoxyviolacein) and crude deox-yviolacein (only deoxyviolacein) produced by the geneticallyengineered strains C. freundii (pComvio) and (pComvioΔd),respectively, were adjusted to an Abs572 or Abs560 of 1.0.Samples in a 48-well plate were treated by exposure to a 30-W UV lamp at 30 cm (400 μW cm−2) or natural light for 0,1.5, 3, 4.5, and 6 h, and then the Abs572 or Abs560 wasdetermined with a computer-operated multi-well plate reader.

For the acid/alkali resistance determination, HCl or NaOHwas added to samples of crude Vio and deoxyviolacein toproduce final HCl concentrations of 2, 1, 0.5, 0.4, 0.3, 0.2,10−1, 10−2, 10−3, and 10−4 N or concentrations of 0.2, 10−1,10−2, 10−3, and 10−4 N for NaOH. A full spectrum scan wasused to check for changes in Vio and deoxyviolacein.

Inhibition of deoxyviolacein on microorganisms in vitro

After the deoxyviolacein was separated and purified fromthe culture, the antimicrobial effect of deoxyviolacein wasexamined using various bacteria (Bacillus licheniformis,Bacillus subtilis, Pseudomonas putida, E. coli, and Enter-obacter aerogenes). The incubation experiment for the an-tibacterial action of deoxyviolacein was carried out byadding 200 μg mL−1 deoxyviolacein to the culture that wereinitiated with 1% (v/v) of an inoculum of incubated over-night cells in the LB medium containing 5 gL−1 yeastextract, 10 gL−1 tryptone, and 10 gL−1 NaCl. Incubationtemperature and time were 30°C for 24 h, respectively. The

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incubation experiment for the antibacterial action of200 μg mL−1 Vio was carried out as the control.

Cytotoxicity assay of deoxyviolacein

Violacein and deoxyviolacein were dissolved in DMSO anddiluted to the indicated concentrations before experimentswith DMEM, supplemented as described above, and thefinal DMSO concentration did not exceed 0.01% (v/v)in any experiments. HepG2 cell viability was assessed by aMTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazoliumbromide) reduction assay, as previously reported (Carmichaelet al. 1987). Briefly, HepG2 cells were seeded into 96-wellplates, followed by an MTT assay. HepG2 were seeded into96-well culture plates at a viable cell density of 5×103

cells/well. After 24 h, the medium was changed toDMEM containing 0.5% NCS for 48 h to synchronizethe cell cycle stage; the blank sample contained onlyDMEM with 0.5% NCS and the control samplecontained DMEM with 0.5% NCS and HepG2 cells.Test samples containing DMEM with 0.5% NCS andHepG2 cells were treated with Vio or deoxyviolacein in0.1% DMSO at various concentrations (0.01, 0.1, 1, 5,and 10 μmol L−1). All samples were incubated for 24and 48 h and then a 200-μL volume of 0.5 mg mL−1

MTT in DMEM was added to each well, and incubatedfor 4 h at 37°C. The cell culture fluid was removed and150 μL of DMSO was added to each well to dissolvethe formazan crystals. After 15 min of incubation, theplate was shaken lightly to facilitate formazan dissolutionand the Abs570 was measured with the multi-scanningspectrophotometer.

Results

Production of deoxyviolacein by recombinant C. freundii(pComvioΔd) using SOE-PCR-mediated viod gene disruption

Prior to this work, a wild or genetically engineereddeoxyviolacein-producing strain was unavailable. Here, theentire viod gene of Vio gene cluster was deleted successfullyto produce a deleted mutant pComvioΔd (SupplementalFig. 1). C. freundii (pComvioΔd) was confirmed both byDNA sequencing and by production of a purple pigment,thereby establishing, for the first time, a recombinant strainproducing only deoxyviolacein.

The vioabce-harboring transformant of C. freundii withpComvioΔd could excrete deoxyviolacein into the mediumas well as form much darker than normal purple colonies at30°C after a 12-h cultivation, indicating stronger expressionof the vioΔd gene cluster in C. freundii than the vio genecluster, which forms dark blue colonies after at least an 18-h cultivation. It was interesting that the deoxyviolacein-

producing recombinant C. freundii (pComvioΔd) showedstrong heterologous deoxyviolacein production ability.

The expression profile of the proteins responsible fordeoxyviolacein production in C. freundii (pComvioΔd)was analyzed by SDS-PAGE. No obvious heterologousVio protein expression band was detected in the strains ofC. freundii (pComvioΔd) (data not shown), which was thesame as Vio-producing C. freundii (pComvio) (Jiang et al.2010). This indicated that the proteins responsible for deox-yviolacein production showed high activities in C. freundii,and it is possible that intracellular physiological conditionsof C. freundii, such as intracellular pH, will play importantroles for the Vio enzyme activities.

Composition and chemical structure analysisof fermentation extracts from genetically engineered strainsof C. freundii (pComvioΔd)

The ethanol extracts of pigment from a C. freundii (pCom-vioΔd) culture was diluted with ethanol to Abs560 1.0 andanalyzed by HPLC. The single peak presented in the elutionprofile in HPLC indicated that the recombinant C. freundii(pComvioΔd) only produced deoxyviolacein (Fig. 2), andthe pigments produced by this and the C. freundii (pCom-vio) strain were spectroscopically identical to deoxyviolaceinproduced by cultures of original strain Duganella sp. B2 andrecombinant C. freundii (pComvio), respectively (Wang et al.2009; Jiang et al. 2010). Due to deoxyviolacein’s insolubilityin water, the extract from C. freundii (pComvioΔd) exhibitedhigh purity according to HPLC analysis, with deoxyviolaceincontent >99%; the parameters for HPLC analysis and productcharacterization were described in the “Materials and methods”section. These results indicated that the VioD enzyme strictlyregulated Vio synthesis and viod gene disruption completelyabolished Vio production.

It is important to note that the disruption of viod led tonew phenotypes which varied in color. The disruption ofviod retained violet pigment production, which was differentfrom the blue pigment appearance of the crude Vio (Fig. 2a).

The molecular masses (m/z) of this component was de-termined as 328.23 (positive) by using HPLC-MS (Fig. 2b),which was consistent with the loss of an oxygen atom fromVio produced by Duganella sp. B2 (Supplemental Fig. 2)(Wang et al. 2009). The 1H–13C-NMR spectroscopy of thiscomponent were compared with the reported data for thebacterial deoxyviolacein as shown in Table 3. The 1H–13C-NMR signals of the pigment agreed very closely with thoseof the reported deoxyviolacein. According to these analyti-cal results above and the previously published publications(Tsutomu et al. 1987; Nakamura et al. 2002), the pigmentproduced from C. freundii (pComvioΔd) was estimated tobe deoxyviolacein (Mw0327). The structure of the deoxy-violacein was shown in Fig. 1.

1526 Appl Microbiol Biotechnol (2012) 94:1521–1532

A)

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Mix #791 RT: 10.75 AV: 1 NL: 1.71E5T: + c d Full ms2 328.58@35.00 [ 80.00-670.00]

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Fig. 2 a HPLC chromatograms of the culture extracts from wild strainDuganella sp. B2, genetic strains of C. freundii (pComvio), and C.freundii (pComvioΔd). b UV–visible and mass spectra ofdeoxyviolacein-producing C. freundii (pComvioΔd). To avoid as muchas possible the repetition of data with our previous report (Wang et al.

2009; Jiang et al. 2010), the UV/Vis spectra and MS of Vio anddeoxyviolacein isolated from crude Vio derived from Duganella sp.B2 and genetic strains of C. freundii (pComvio) were not shown here.However, for each of the components, the characteristic MS and UV/Vis spectra were confirmed

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Fermentation characteristics of C. freundii (pComvioΔd)

Aerobic batch fermentations were performed with glycerolas the carbon source and L-tryptophan as the Vio precursor.Analysis of time course profiles of cell growth and deoxy-violacein production by recombinant C. freundii (pCom-vioΔd) showed that violet deoxyviolacein was detectablefrom the beginning of cell growth and reached maximumconcentrations in the late stationary phase which showedstable deoxyviolacein production. This pattern was the sameas that of C. freundii (pComvio).

Deoxyviolacein was produced after the cultivation temper-ature was shifted to 20°C and, after cell growth slowed down,deoxyviolacein production increased and reached a maximumconcentration of 1.9 gL−1 after an incubation time of 44 h(Fig. 3). Meanwhile, the maximum deoxyviolacein produc-tivity was as high as 52.1 mg L−1 h−1, which was higher thanthat of crude Vio produced by C. freundii (pComvio) and wasto date the first report of pure deoxyviolacein production.Deoxyviolacein produced by the recombinant C. freundiicould be rapidly and completely extracted from the cell pelletsusing ethanol without requiring any other cell breakage

procedure, indicating that deoxyviolacein is extracellular se-cretion, like the Vio production system.

Real-time RT-PCR analysis of vioe gene transcriptionin recombinant C. freundii strains

vioe was selected to detect its expression ratio due to vioe isthe last gene in the Vio biosynthesis gene cluster and could

Table 3 1H–13C-NMR analysisof the purified pigment andthe comparison with the reporteddata

1H-NMR 13C-NMR

Position Sample resource

Nakamuraet al. (2002)

Tsutomuet al. (1987)

C. freundii(pComvioΔd)

Nakamuraet al. (2002)

Tsutomuet al. (1987)

C. freundii(pComvioΔd)

1 12.13 12.20

2 8.19 8.21 8.18 129.3 131.0 129.4

3 106.3 106.3

4 124.4 124.5

5 7.85 7.88 7.85 119.7 120.0 119.8

6 123.1 123.2

7 7.30 7.31 7.31 121.5 122.5 121.6

8 7.56 7.59 7.56 112.9 113.5 113.0

9 137.4 137.4

10 10.83 10.84 10.80

11 171.5 171.5

12 136.7 136.8

13 7.68 7.68 7.65 97.4 97.5 97.4

14 147.0 147.0

15 10.67 10.64 10.63

16 170.1 170.2

17 119.5 119.6

18 122.2 122.3

19 8.96 8.95 8.95 126.4 126.8 126.5

20 6.96 6.96 6.96 120.9 121.2 121.0

21 7.22 7.22 7.22 129.7 129.7 129.8

22 6.83 6.85 6.84 109.1 109.0 109.1

23 141.9 141.9

Fig. 3 Fermentation characteristics of C. freundii (pComvioΔd). Datarepresent the means ± SD of three replicates

1528 Appl Microbiol Biotechnol (2012) 94:1521–1532

present the expression ratio of Vio gene cluster. The expres-sion of vioe in different recombinant C. freundii strains wasdemonstrated by detection of the corresponding mRNAusing real-time RT-PCR. The absence of viod appeared tohave no effect on cell growth of C. freundii (pComvioΔd)(Fig. 3) when compared to C. freundii (pComvio) (Jiang etal. 2010). As gene expression often varied significantly indifferent growth phases, all cells used for RNA isolationwere in the logarithmic phase to minimize this effect on RT-PCR analysis. Using 16S rRNA as the internal standard, itwas clearly observed that the vioe gene transcription level inviod-deleted C. freundii (pComvioΔd) was about 2.2-foldgreater than in the Vio-synthesizing strain C. freundii(pComvio) at the same culture conditions, which indicatedthat the gene organization of viod disruptions could possiblypotentially induce polar effects on the downstream vioe genewithin this small operon (Supplemental Fig. 3). However,the increase in Vio enzyme activities was not proportional tothe vioe transcription rate.

Acid/alkali and UV resistance of deoxyviolacein

The resistance of Vio and deoxyviolacein to acid/alkaliconditions was initially determined by visual color compar-isons. Supplemental Fig. 5 illustrates the color changes ofVio and deoxyviolacein. When >10−1 N NaOH was addedto the deoxyviolacein ethanol solution, the original blue–purple color visibly became weak, quickly diminished, andthen turned to green, while, in contrast, Vio’s blue–purplecolor quickly turned to green when 10−1 N NaOH wasadded to the ethanol solution of Vio. The full spectrum scans(Fig. 4a) showed that, in the range of [HCl] from 10−4 to 2 Nand the range of [OH−] from 10−4 to 10−1 N, deoxyviolaceinexhibited maximum absorption, with a relative absorbanceof 1.0, while the Vio maximum absorption significantlydecreased under the acid conditions of >0.3 N HCl or alkaliconditions of >10−2 N NaOH.

Examination of the photostability of Vio and deoxyvio-lacein against UV/Vis radiation, isothermally at room

A)

Wavelength (nm)300 400 500 600 700 800 900 1000 300 400 500 600 700 800 900 1000

OD

val

ue (

Vio

lace

in)

0.0

.2

.4

.6

.8

1.0

0.0

.2

.4

.6

.8

1.0HCl 2N HCl 1N HCl 0.5N HCl 0.4N HCl 0.3N HCl 0.2N HCl 0.1N HCl 0.01N HCl 0.001N HCl 0.0001N CONTROL NaOH 0.0001NNaOH 0.001N NaOH 0.01N NaOH 0.1N NaOH 0.2N

Wavelength (nm)

OD

val

ue (

Deo

xyvi

olac

ein) HCl 2N

HCl 1N HCl 0.5N HCl 0.4N HCl 0.3N HCl 0.2N HCl 0.1N HCl 0.01N HCl 0.001N HCl 0.0001N CONTROL NaOH 0.0001NNaOH 0.001N NaOH 0.01N NaOH 0.1N NaOH 0.2N

B)

0 1 2 3 4 5 6 7

Abs

orpt

ance

.4

.6

.8

1.0

UV-Violacein

UV-DeoxyviolaceinNatural light-Violacein

Natural light-Deoxyviolacein

( )

Fig. 4 a Full spectrum absorption of Vio and deoxyviolacein under different acid/alkali conditions. b UV resistance of Vio and deoxyviolacein.Data represent the means ± SD of triplicates

Appl Microbiol Biotechnol (2012) 94:1521–1532 1529

temperature, showed changes in maximum absorption ver-sus exposure time (Fig. 4b). After 6 h of UV treatment, Vioabsorbance decreased much more than that of deoxyviola-cein, indicating UV degradation. And, interestingly, deoxy-violacein showed greater stability than Vio after 6 h ofexposure to natural light. Thus, it was concluded that deox-yviolacein possessed better photostability and acid/alkaliresistance than Vio.

Antibacterial activity of deoxyviolacein

Figure 5 shows the growth inhibitory effect of deoxyviolaceinon two Gram-positive B. licheniformis and B. subtilis andthree Gram-negative bacteria of E. coli, P. pseudomonas,and E. aerogenes after the addition of deoxyviolacein in theearly phase of cell growth; 200 μg mL−1 deoxyviolaceinshowed the remarkable antibacterial activity for Gram-positive bacteria such as B. licheniformis and B. subtilis, andalmost 80% of cells growth was inhibited after an incubationtime of 24 h. However, it could not inhibit the growth ofGram-negative bacteria such as E. coli even if more than500 μg mL−1 deoxyviolacein was added (data not shown).Though the reason why the psychrotrophic bacterium produ-ces two kind of violet pigments, i.e., violacein and deoxyvio-lacein, is not known, it may be considered that theantibacterial activity for the putrefactive bacteria is increasedby the additive effects obtained by the two components.

Growth and proliferation inhibition on hepatocellularcarcinoma cells (HepG2) of deoxyviolacein

HepG2 were treated with 0.1–10 μM concentrations of Vio ordeoxyviolacein for 24–48 h and the effects assessed in termsof viability and cell proliferation. The results indicated that

cell treatment with Vio or deoxyviolacein at concentrations<10 μM for 24–48 h produced significant inhibition in cellviability, compared with controls (Fig. 6). Deoxyviolaceinwas found to inhibit HepG2 cell proliferation at low concen-trations of 0.1–1 μM. However, the inhibition effects keepconstant with increased concentrations. In comparison, Viowas found to remarkably inhibit HepG2 cell proliferation in adose-dependent manner between 0.1 and 10 μM (Fig. 6).

Discussion

Deoxyviolacein is the deoxy-analog of Vio, having oneless oxygen atom at the 2-position of the indole ring, and

E.aerogenes E.coli P.putida B.subtilis B.licheniformis0.0

0.4

0.8

1.2

1.6

2.0

OD

660

Strains

ck Violacein Deoxyviolacein

Fig. 5 Inhibition of Vio and deoxyviolacein on cell growth in bacteriafor 24 h. Data represent the means ± SD of three replicates

Fig. 6 Effect of crude Vio and deoxyviolacein on HepG2 cell viability.HepG2 cells incubated with different concentrations of Vio and deox-yviolacein (0–10 μM) at 24 (open columns) and 48 h (filled columns);living HepG2 cellular activity was assessed by MTT assay; *p<0.01compared to control cells (ANOVA, Tukey test); results representmeans ± SD of three experiments run in sextuplicate

1530 Appl Microbiol Biotechnol (2012) 94:1521–1532

occurs naturally as a minor component in the wild-typeVio-producing bacterial strains (Sanchez et al. 2006). TheVioD enzyme has been shown to be responsible for theaddition of an oxygen atom on the Vio oxindole subunit(Sanchez et al. 2006).

In this study, a simple and fast SOE-PCR method wasused to produce a viod gene-knockout construct, without theuse of DNA ligase and plasmid vectors, but instead involv-ing only a two-step PCR, including one standard PCR andone fusion PCR. This method could be used in the field ofmetabolic engineering as a fast and versatile tool for design-ing an exact gene-knockout construct, without the introduc-tion of extra bases, by choosing primer binding sites at thetarget locus. Furthermore, the C. freundii-based heterolo-gous Vio biosynthetic system provides a suitable platformfor fast and simple, rational redesigning of biosyntheticpathways for deoxyviolacein production. In the presentcase, a recombinant plasmid, which encoded the genescapable of only efficiently synthesizing deoxyviolacein,was successfully constructed and introduced into C. freundii,and the deletion of the viod gene led to the absence of ahydroxyl group at the 2-position of the Vio indole ring. Noobvious biosynthetic intermediate accumulation was observedwhen the viod gene was deleted from the Vio biosyntheticpathway, which highlighted the essential nature of the presentdesign of the Vio biosynthesis pathway for obtaining puredeoxyviolacein. These results further indicated the rationale ofthe Vio biosynthesis pathway.

Although the present results showed that the productionkinetics of deoxyviolacein by C. freundii (pComvioΔd) wasconsistent with that of Vio by C. freundii (pComvio), the fer-mentation conditions were different, such that only 0.4 gL−1 ofdeoxyviolacein was synthesized with the optimum E2-Vio me-dium for our previous Vio-producing C. freundii (pComvio)(Supplemental Fig. 4), but deoxyviolacein production reached3.9 gL−1 when peptone was used as the organic nitrogen sourcein optimum E2-deoxy medium for C. freundii (pComvioΔd)lacking Fe2+ andMg2+ (unpublished data), which suggested thatVio biosynthesis and deoxyviolacein may be regulated by someunknown factors. It is possible that the decreased proportion ofVio in recombinant C. freundii (pComvio) was caused by inef-ficient VioD activity (Sanchez et al. 2006). Furthermore, as itwould be promising to produce pure Vio by controlling the viogene(s) and fermentation conditions, this goal is currently understudy.

Here, the physical characteristics of crude Vio and deoxy-violacein and their possible inhibitory effects on tumor cellswere examined. Deoxyviolacein’s structure differed from Vioonly in the oxygen atom at the Vio indole ring 2-position;however, there were significant differences in the maximumabsorbance peaks and physical properties, such as the colorand acid/alkali tolerance, between these two compounds. Theinfluence of organic chemical structure on their absorption

maxima and spectral shape is well known, and the UV/Visspectrum of a pigment is of great importance to a compound’shue. The hydroxyl group bonded to the Vio indole ring influ-enced the conjugated systems, shifting the absorbance peaksto longer wavelengths (572 nm) than the peak wavelength ofdeoxyviolacein (560 nm). Thus, the hydroxyl group influ-enced the color and acid/alkali tolerance of Vio; deoxyviola-cein has a slight purple color whereas Vio is blue (Fig. 2).

The results from this study showed that deoxyviolaceinpossessed functional similarity to Vio. However, the ab-sence of a hydroxyl group at position C-2 strongly de-creased the antitumor activity. It is possible that thepresence or absence of the hydroxyl group affected thephysical properties of Vio and deoxyviolacein, respectively,ultimately leading to different antitumor efficacies. Thus,Vio was a more active antitumor agent.

In summary, genetically engineered C. freundii with viogene cluster or a viod-deleted vio gene cluster could bepromising tools that can help in further characterization ofthe Vio biosynthetic pathway as well as the related regula-tory mechanisms and in clarifying the physiological reasonwhy deoxyviolacein always accompanies Vio production.Furthermore, these studies shed light on correlations be-tween the chemical structures of Vio-type molecules andtheir bioactivity profiles.

Acknowledgments This work was supported in part by the NationalScience Fund of China (Grant no. 21006058), a China PostdoctoralScience Foundation funded project (Grant no. 20080430367), a BasicResearch Fund of CAAS (0042009001), and a Xinjiang-supportingproject by Science and Technology (Grant no. 200991132).

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