Augmented production of poly-β- d-mannuronate and its acetylated forms by Pseudomonas

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Augmented production of poly-β- d-mannuronate and its acetylated forms by Pseudomonas

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  Process Biochemistry 46 (2011) 328–334 Contents lists available at ScienceDirect Process Biochemistry  journal homepage: www.elsevier.com/locate/procbio Augmented production of poly-  - d -mannuronate and its acetylated formsby  Pseudomonas Yaligara Veeranagouda a , Chitragara Basavaraja b , Hyun-Sook Bae c , Kwang-Hyeon Liu d , Kyoung Lee a , ∗ a Department of Microbiology, Changwon National University, Changwon, Kyongnam 641-773, Republic of Korea b Department of Chemistry, Institute of Functional Materials, Inje University, Kimhae, Kyungnam 621-749, Republic of Korea c Department of Clothing & Textiles, Changwon National University, Changwon, Kyongnam 641-773, Republic of Korea d Department of Pharmacology, Inje University, College of Medicine, Busan 614-736, Republic of Korea a r t i c l e i n f o  Article history: Received 16 February 2010Received in revised form 25 August 2010Accepted 14 September 2010 Keywords: Poly-  - d -mannuronatePolymannuronateAlginate Pseudomonas Polymannan alg   gene a b s t r a c t Poly-  - d -mannuronate(PM)anditsderivativeshavepotentialtobeusedinpharmaceuticalapplications.Aspontaneousmutant(E1)of  Pseudomonasalkylphenolia KL28producedlargeamountsofahighlyviscouspolymer consisting of repetitive units of    - d -mannuronic acid with acetylation at the 2nd and/or 3rdcarbon(AcPM).The alg  genecluster(18,275bpwith12ORFs)isessentialfortheproductionofAcPMandhas been sequenced. Mutations in the E1 strain were found in both the  mucA  (resulting in a truncatedprotein) and the  algG  (non-synonymous amino acid substitution in the conserved epimerase domainof mannuronan C-5-epimerase) genes. Disruption of   algI   (acetylase) resulted in the production of a de-acetylatedpolymer,PM.Themolecularweight(viscosity)andtheAcPMproductionlevelwereinfluencedby the addition of NaCl into the culture media. Under optimized flask culture conditions, 8–14g/L of AcPM with different viscosities could be produced. In addition, some rheological properties of the highmolecular weight AcPM produced by the E1 mutant were better suited for pharmaceutical use thancommercialalginate.Inconclusion, P. alkylphenolia E1andits algI  mutantmaybesuitablecandidatesformass production of AcPM and PM, respectively.© 2010 Elsevier Ltd. All rights reserved. 1. Introduction Biopolymers with defined chemical compositions are of spe-cial interest in biomedical research. Poly-  - d -mannuronate (PM)is a component of alginate and is relevant to many medical andpharmaceutical applications. For example, PM and its degrada-tion products activate immune cells, resulting in the secretionof cytokines such as tumor necrosis factor   , interleukin-1 andinterleukin-6, and thus PM can be used to increase immune pro-tection against various infections [1–3]. Sulfated PM has been shown to significantly inhibit HIV-1 replication both  in vitro  and in vivo  [4]. Recent studies demonstrate that oligomannuronatesare highly effective tumor angiogenesis and metastasis inhibitorsand are promising candidates for cancer therapy [5]. Matrix tablets prepared from highly viscous alginate (rich in mannuronicacid) demonstrated an enhanced drug release rate in their acidicphase [6]. It has been recently demonstrated the application of PM in nanocomposite synthesis, in which newly synthesizedpolyaniline–PMnanocompositeshadenhancedconductiveproper-tiescomparedtopolyanilinenanocomposites[7,8].Theexploration ∗ Corresponding author. Tel.: +82 55 213 3486; fax: +82 55 213 3480. E-mail address:  kyounglee@changwon.ac.kr (K. Lee). of novel uses for PM in various medical and pharmaceutical fieldswill likely lead to an increased demand for its production.The PMs in the aforementioned applications are mainly sup-plied from seaweed algae alginate consisting of    - d -mannuronicacid and  - l -guluronic acid, which are linked by a  -1,4 glycosidicbond with varying compositions and sequential structures [9,10].However,concernshavebeenraisedregardingtheuseofPMsfromseaweed in biomedical research because algal alginate is suscep-tible to composition variation due to uncontrolled environmentalfactors [11]. Moreover, extraction of PM from algal alginate can have low purity and high production costs. Thus, there is a grow-ing interest in the production of bacterial PM where qualities suchas monomer composition, polymer chain length and the degree of acetylation can be manipulated [12]. Thus far, two bacterial gen- era,  Azotobacter  and Pseudomonas ,havebeenshowntoproducePMas a component of alginate. Alginate produced by  Azotobacter   andbrown algae contains a higher proportion of   l -guluronic acid thanthatof  Pseudomonas .Althoughafew Pseudomonas strains,including Pseudomonas aeruginosa, produceacetylatedPM(AcPM)withlittleor no guluronic acid content [9,13,14], studies on the efficient pro- duction of PM and AcPM in bacterial hosts are lacking. In addition,culture conditions that affect the molecular sizes of PM and AcPMhave not been thoroughly investigated. In this study, the effect of culture conditions on AcPM productivity and polymer properties 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.procbio.2010.09.009  Y. Veeranagouda et al. / Process Biochemistry 46 (2011) 328–334  329 fromahyperproducerof  Pseudomonasalkylphenolia wasexamined,and genes related to AcPM production were also characterized. 2. Materials and methods  2.1. Bacterial strains, media and culture conditionsP. alkylphenolia  KL28 was previously described as an  n -alkylphenol-degradingbacterium [15,16]. A spontaneous mutant of strain KL28 (E1) that formed a mucoid colony under conditions used for the induction of aerial structures by  p -cresol [16]was used in this study and maintained on LB medium. Recombinant  Escherichiacoli  and  Pseudomonas  strains were treated with antibiotics when necessary usingconcentrations as described previously [17]. Modified King’s B medium (PG) con- sisting of 20g/L of peptone, 10g/L of glycerol and 1.5g/L of MgSO 4 · 7H 2 O in 50mMpotassiumphosphatebuffer(pH6.0)wasusedfortheproductionofAcPMorPMby Pseudomonas .ToassesstheeffectofpHonAcPMproduction,thepHofthemediumwas adjusted with 1M NaOH or 1M HCl. The medium was sterilized by autoclav-ing at 15lb for 20min. Seed cultures were prepared by scraping E1 cells from PGplates grown overnight and suspending them in saline. The absorbance of the cellsuspensionwassetto6.0at660nm.Then,50  Lofthecellsuspensionwasaddedto250-mL Erlenmeyer flasks containing 50mL of medium. The flasks were incubatedat30 ◦ Conarotaryshaker(160rpm),and1mLofthesamplewaswithdrawnatthespecifiedtimeintervals.Growthwasestimatedbymeasuringtheabsorbanceofthecellsuspensionat660nmwithaspectrophotometer(Model2130,SincoCo.,Korea).CellswereremovedbycentrifugationandculturesupernatantswereappropriatelydilutedwithdistilledwatertomeasuretheAcPMproduction,andtheAcPMcontentwas estimated as detailed below.  2.2. Sequencing of the alg gene cluster and mucA gene The  alg   gene cluster of the KL28 strain was probed by homologous recombi-nation using a suicide vector. The  alg   genes retrieved from the junction plasmidswere cloned and sequenced. To construct a suicide vector that cannot replicatein  Pseudomonas , pRL27 [18] was first digested with  Eco RI and the transposon-free fragment was self-ligated. The resulting plasmid was named pRL27  Tnp. TheDNA sequence (1558bp) upstream of the alginate gene cluster and a small por-tion of the 5 ′ -terminus of   algD  were PCR-amplified using the  P. alkylphenolia  KL28chromosome as a template. Primers E1-algDpF (5 ′ -CGACGACCTGCTGCTCAACC-3 ′ )and E1-algDpR (5 ′ -ATCGGTGGCTGGTCGTAA-3 ′ ) were used. These primers weredesigned based on conserved sequences from  Pseudomonas alg   gene clusters. Theamplified PCR product was purified and ligated into pGEM ® -T Easy vector, yield-ing pT-algP. The presence of the intact, amplified,  alg   sequence in pT-algP wasconfirmed by DNA sequencing. The  Eco RI fragment from pT-algP containing theamplified gene was cloned into the suicide vector pRL27  Tnp. The resulting plas-midwasnamedpRL27  Tnp-algP(Fig.S1inSupportingInformation)andwasused in a homologous recombination reaction with the  alg   cluster of KL28. A singlecrossover mutant of the  alg   gene was created through a triple mating between  P.alkylphenolia  KL28,  E. coli  DH5   (pRL27  Tnp-algP), and  E. coli  DH5   (pRK2013)[19]. Mutants were selected on LB agar containing ampicillin and kanamicin andconfirmed by PCR. Chromosomal DNA from the mutants was digested with  Bam HIand self-ligated to recover the whole  alg   gene cluster containing the suicide plas-mid. A 22-kb junction plasmid obtained from the above procedure was sequencedat SolGent Co. Ltd. (Taejeon, Korea) using an automated sequencing apparatus(ABI PRISM 377, PE Biosystems Inc.). ORFs were identified using the GETORF pro-gram (http://bioweb.pasteur.fr/seqanal/interfaces/getorf.html, Pasteur Institute).The deduced amino acid sequences were compared with the protein sequencedatabase (GenBank) using the BLASTX algorithm (http://www.ncbi.nlm.nih.gov).Thenucleotidesequenceofthe alg  geneclusterwasdepositedintheNCBInucleotidesequence database under the accession number GU597845.The  mucA  gene was PCR-amplified with primers MucAF (5 ′ -GAGTTT-TACGACGGCGATCATGG-3 ′ ) and MucAR (5 ′ -GCTCACACCAGACACCAAGGCC-3 ′ ) withKL28 and E1 chromosomes. The amplified PCR product (1.1kb) was purified andsubjectedtonucleotidesequenceanalysis.Thenucleotidesequenceof  mucA anditsflanking genes was deposited in the NCBI nucleotide sequence database under theaccession number HM172485.  2.3. Measurement of P  algD -gfp gene expression Theamplified Eco RIfragmentfromthepT-algPvectorwasclonedintothesamerestriction site of the GFP reporter vector pPROBE-GT to assess  alg   promoter activ-ity [20]. The resulting plasmid with the correct promoter orientation was called pPROBE-GT-algP (Fig. S1 in Supporting Information) and complemented the KL28 andE1strains.Tomeasure alg  promoteractivity(P algD ),strainscontainingpPROBE-GT-algP were grown in PG liquid medium with gentamicin under the conditionsdescribed above. At specified time points, 1-mL aliquots were harvested by cen-trifugation,washedtwicewithsaline,andresuspendedinsalineatanOD 660  of  ∼ 0.4.The GFP-expression level was measured using a spectrofluorophotometer (ModelRF-5391PC, Shimadza Co.) as previously described with excitation and emissionwavelengths of 450 and 509, respectively, with 3.0nm wavelength splits [21].  2.4. Generation of an algI non-polar mutant  Atetracycline-resistant(Tc R  )cassettewithoutatranscriptional-terminatorwasconstructed as follows. First, the Tc R  cassette was amplified from p34S-Tc [22]with primers Tc-F1 (5 ′ -CCGAGCTCATGTTTGACAGCTTATC-3 ′ ,  Sac  I restriction siteunderlined) and Tc-R1 (5 ′ -TGCCGCCGAGCTCCATTCAGGTCG-3 ′ ,  Sac  I restriction siteunderlined).TheamplifiedfragmentwasclonedintopGEM ® -TEasyvectorbyselect-ingtetracycline-resistant E.coli DH5  recombinants.TheintactnucleotidesequencewasalsoconfirmedbyDNAsequencing.FromtheresultingvectorpGEM-Teasy–Tc2,the new Tc R  cassette (Tc2) from the  Sac  I digestion was cloned into p34S-Tc wherethe srcinal Tc R  gene was removed by  Sac  I digestion. The resulting plasmid wasnamed p34S-Tc2 (Fig. S1 in Supporting Information).A non-polar E1  algI   knockout mutant was generated by insertional muta-genesis with the Tc2 cassette, followed by allelic replacement as described bySchafer et al. [23]. Briefly, a 1.1-kb fragment including parts of the  algI   and algJ   genes was PCR-amplified using the  P. alkylphenolia  KL28 chromosome as atemplate. The primers AlgIF1 (5 ′ -CCGACCATTGCTTCGCCCTACAGA-3 ′ ) and AlgJR1(5 ′ -GCATGCTCGTCGTGAACAGCCACT-3 ′ )wereused.Theamplifiedproductwaspuri-fied, ligated into pGEM ® -T Easy vector, and named pT-algI-J. The presence of an intact amplified gene sequence in pT-algI-J was confirmed by DNA sequenc-ing. The  Eco RI– Sph I fragment from pT-algI-J was cloned into pK18 mobsacB  [23]to yield pK18 mobsacB -algI-J. The Tc2 cassette from p34S-Tc2 was cloned into theunique  Sac  I site of   algI   in pK18 mobsacB -algI-J to disrupt the gene. The resultingplasmid was called pK18 mobsacB -algI-J-Tc2 (Fig. S1 in Supporting Information)and used to disrupt the  algI   gene in E1 via a triple mating as described previ-ously [17]. The E1( algI  ::Tc2) mutant was selected as described previously, exceptthat the LB agar was supplemented with tetracycline [23].  algI   disruption inE1( algI  ::Tc2) was further confirmed by the sequencing of the PCR product obtainedusing the primers AlgIFa (5 ′ -CGACCTGTGCATCCTCGGCTATTTC-3 ′ ) and TcIR (5 ′ -TGCGACTCCTGCATTAGGAAGC-3 ′ ) with template DNA from E1( algI  ::Tc2). The twoprimers bind upstream of the AlgIF1-binding sequence and the Tc R  gene, respec-tively.  2.5. Construction of an algG expression vector and complementation in E1 The  algG  expression vector, pAlgG was constructed as follows. The  algG  genewas PCR-amplified with primers AlgGF (5 ′ -CCGGATCCTTGAGCCAGTCGGTCGA-3 ′ , Bam HI site underlined) and AlgGR (5 ′ -AAGGCAGAGCTCAGGCCCAGCAGTT-3 ′ ,  Sac  Isite underlined) with the KL28 chromosome as a template. The 1.8-kb productwas purified and ligated into pGEM ® -T Easy (Promega Co.), yielding pT - AlgG. Thepresence of an intact  algG  gene in pT-AlgG was confirmed by DNA sequencing.The amplified  Bam HI –Sac  I fragment from pT-AlgG was ligated into the same sitesof pBBR1MCS-5, which has a broad host range [24]. The resulting pAlgG plasmid (Fig. S1 in Supporting Information) was transformed into strain E1 by tri-parental mating to yield the E1(pAlgG) strain.  2.6. Preparation and quantification of polymers for analysis The AcPM and PM contents in the culture supernatants were measured by the m -hydroxybiphenylmethodaspreviouslydescribedwithpurifiedAcPMandPMasstandards [25]. To obtain the large amount of AcPM and PM required for various measurements, culture broths were centrifuged at 12,000rpm for 1h at 4 ◦ C. Whenthe culture broths were too viscous, they were diluted with saline and centrifuged.AcPM and PM were precipitated by adding equal volumes of isopropyl alcohol andremoved with a glass rod. The precipitate was dissolved in distilled water and pre-cipitatedwiththreevolumesofethanol.TheresultingPMwaswashedseveraltimeswith 100% ethanol and dried at 80 ◦ C to a constant weight.  2.7. HPLC analyses to determine the sugar acid composition and molecular weight of the polymer  The sugar acid compositions of AcPM and PM were analyzed with trifluo-roaceticacidhydrolysatesbyahigh-performanceanion-exchangechromatographywith pulsed amperometric detection system (Dionex, Sunnyvale, CA, USA) usinga CarboPac TM PA-1 column as previously described [26]. The molecular weight of  AcPMwasmeasuredbyaWatersGPCsystemequippedwithaPLAquagel-OHmixedcolumn (Polymer Laboratories Ltd., UK, 8  m, 7.8mm × 300mm) and a Waters 515HPLCpump.SampleselutedfromthecolumnweredetectedwithaWaters2410dif-ferential refractometer. AcPM samples were filtered through a 0.22-  M Milliporemembrane to remove microgels and a 100-  L sample (3mg/mL) was subsequentlyinjected into the GPC system. The AcPM was eluted using 0.2M NaNO 3  and 10mMNaH 2 PO 4  (pH7.0)at35 ◦ Cwithaflowrateof1mL/min.Pullulansof11,800,22,800,47,300, 112,000, 212,000, 404,000 and 788,000Da were used as standards. Themolecular weight of AcPM was calculated based on the retention times of the stan-dards and AcPM. This experiment was carried out by the Korea Polymer Testing &Research Institute, Seoul.  330  Y. Veeranagouda et al. / Process Biochemistry 46 (2011) 328–334 Fig. 1.  Polymer produced by the E1 strain. Polymer produced from 500mL of E1culture supernatant was recovered using a glass stirring rod following isopropylalcohol precipitation. Petri dish size: Ø 100 ×  h  20mm.  2.8.  1 H NMR determinations The AcPM and PM obtained from the procedure described above were partiallyacid hydrolyzed to avoid line broadening caused by polymer viscosity. In brief, theAcPMandPMweredissolvedinwateranddialyzedagainsttripledistilledwaterfor48h, and the PM was recovered by isopropyl alcohol precipitation. About 400mgof this sample was dissolved in 20mL of distilled water and the pH of the solutionwasadjustedto3.0using1MHCl.Thesolutionwasthenboiledfor1handneutral-ized to pH 7.0 with 1M NaOH. The resulting solution was dialyzed against 10L of distilled water for 48h and concentrated by freeze-drying. Then, 20mg of the acid-hydrolyzed polymers were dissolved in 1mL of D 2 O and the spectra were recordedat 82 ◦ C. All of the NMR experiments were performed on a Bruker spectrometer at400.13MHzfor 1 Hnuclei.Thefollowingacquisitionparameterswereused:numberofscans=32;pulsewidth=9.3  s;numberofpointsintimedomain=32;inter-pulsedelay=1s; spectral width=6793Hz; acquisition time=2.41s; and line broadeningforexponentialwindowfunction=0.3Hz.ThereferencepeakwasDHOat4.71ppmat 82 ◦ C. The degree of   O -acetylation in AcPM ((number of acetyl groups/uronicacid) × 100) was deduced from the  1 H NMR spectra by comparing the intensitiesof acetyl proton signals ( I  a , at 2.6–2.8) with those of the anomeric proton signal ( I  b ,5.1–5.9)ofmannuronicacidwithacalculationof  I  a /( I  b  − I  a /3)aspreviouslydescribed[13].  2.9. Measurement of PM viscosity The viscosity of culture supernatants and purified AcPM or PM was measuredwith a Vibro Viscometer (SV-10, A & D Company, Japan) at 25 ◦ C with a vibrationfrequency of 30Hz. The influence of temperature on viscosity of 0.25% (w/v) poly-mer solutions was determined at a fixed temperature and after thermal treatment.To measure the influence of pH on viscosity, the pH of aqueous polymer solutions(0.25%, w/v) was adjusted with either 1M HCl or 1M NaOH. The effect of salt onthe viscosity was determined by preparing polymer solutions with different saltconcentrations.ThecontrolsodiumalginatefrombrownalgaewaspurchasedfromAldrich Chemical Co. (No. 180947). 3. Results  3.1. Isolation and identification of the polymer produced by theE1 strain AspontaneousmutantofKL28,designatedE1,whichproducealarge amount of polymer when grown in PG medium was isolatedas described in Materials and methods. The mucoid phenotypeassociated with E1 was stable for more than 100 generations. Thepolymers produced by this strain could be readily recovered byisopropyl alcohol precipitation as described in Section 2 (Fig. 1). Chemical analysis revealed that the polymers were mainly com-posedofuronicacid.Incontrast,thewild-typeKL28strainwasnotmucoidanddidnotproduceanydetectableamountofuronicacid.HPLC analysis of the acid-hydrolyzed E1 polymers revealed a sin- Fig. 2.  HPLC analysis of sugar acid content of the acid-hydrolyzed polymers. gle peak with a similar retention time to the second peak formedbyacid-hydrolyzedcommercialalginate(Fig.2).Becausecommer- cial uronic acid standards for alginate are not currently available,the structure of the E1 polymer was determined using  1 H NMR spectroscopy. The  1 H NMR spectrum of partially acid-hydrolyzedE1-polymers is shown in Fig. 3A. The chemical shift assignment of  the E1 polymers was achieved by comparing previously reportedchemical shifts of AcPM [9]. The acetylation and H1–H5 pro- tons have been assigned as shown in Fig. 3A. No resonance was detected in the spectrum for guluronic acid. H2 ′ (5.58ppm) andH3 ′ (5.96ppm) in the anomeric region were assigned to the H2and H3 protons, respectively, in the acetylated mannuronic acidcomponents [9]. From these results, it was concluded that the E1 polymers were mono-acetylated at the O-2/O-3 position or di-acetylated at both the O-2 and O-3 positions to a degree of 37.4%acetylation.These results were confirmed with de-acetylated PM producedbytheengineeredacetylase-negativemutantE1( algI  ::Tc2).ThePMproduced by the mutant E1 did not show chemical shifts for acetyl Fig. 3.  1 H NMR spectra of the polymers formed by the E1 strain (A) and the  algI  mutant of E1 (B). H2 ′ and H3 ′ are the corresponding H2 and H3 protons in theacetylated components [9].  Y. Veeranagouda et al. / Process Biochemistry 46 (2011) 328–334  331 A algAalgF algJ algI algLalgX algG algE algK alg44alg8 algDP(alg) 937978927987908777789192 483215385485434466521493557388496438aaof Number  aaHighest(%)Identity B Fig. 4.  Organization of the  alg   gene cluster of   P. alkylphenolia  KL28. (A) Arrangement of   alg   genes. (B) Alignment of a portion of the AlgG amino acid sequence. The AlgGaminoacidsequenceswerefromthefollowingsources.KL28, P.alkylphenolia ;PAO1, P.aeruginosa PAO1;PSY, P.syringae pv.syringaeB728a;PfO1, P.fluorescens PfO1;PEL48, P. entomophila  L48; PPF1,  P. putida  F1; KT2440,  P. putida  KT2440; and E1, spontaneous mutant of KL28. The amino acids identical to those of strain KL28 were indicated withdots. The amino acids shown to be essential for the epimerase function of AlgG were boxed. groups but did display five major signals whose chemical shiftswere identical to those of previously reported  1 H chemicals shiftsofPM(Fig.3B)[9].Inaddition,theacid-hydrolyzedpolymerformed by E1( algI  ::Tc2) produced a single peak corresponding to the sec-ond peak of alginate in the HPLC analysis (Fig. 2). Therefore, based on the HPLC and  1 H NMR analyses, it was confirmed that the poly-mersproducedbytheE1andE1( algI  ::Tc2)strainsareAcPMandPM,respectively. In addition, the monomers, mannuronic acid, mono-acetyl mannuronic acid and di-acetyl mannuronic acid obtainedby the acid hydrolysis of those polymers were identified usingelectrospray tandem spectrometry as shown in Fig. S2 (see Sup-porting Information).  3.2. Nucleotide sequence and level of alg gene expression instrains KL28 and E1 The  alg   gene cluster necessary for alginate production wasretrieved from  P. alkylphenolia  KL28 and sequenced as describedin Section 2. The alginate biosynthesis genes of KL28 were organized in a manner similar to other alginate-producing  Pseu-domonas  species [12,27]. Based on sequence homology, the 12 ORFs (18,275bp) were arranged in the same transcriptional ori-entation (Fig. 4A). The proteins from the deduced amino acid sequences had identities of 77–93% to known alginate biosyn-thesis proteins of other  Pseudomonas  species. Expression level of the promoter upstream of   algD  was investigated using a GFP-based reporter assay. The specific GFP expression found in thestrains KL28(pPROBE-GT-algP) and E1(pPROBE-GT-algP) after 48hof culture was 2.5 ± 0.25 and 68 ± 3, respectively. These resultsindicated that the  alg   cluster was strongly expressed only in theE1 strain and were consistent with the observation that AcPMwas not detected from the culture supernatant of wild-type strainKL28.The  mucA  gene product inhibits the function of AlgU, a sigmafactorthatinducestheexpressionof  algD [12].Inmanycases,over-expression of alginate coincides with mutations in  mucA , and forthis reason,  mucA  was sequenced in the E1 and KL28 strains. Inter-estingly, the  mucA  gene in E1 contained a point mutation (C169T)resulting in a stop codon (Fig. S3 in Supporting Information). This shortened MucA (56 amino acid residues of 196 residues) likelydidnotinhibitAlgUwell,andthus,thismutationledtothemucoidphenotype in E1.  3.3. Identification of a point mutation in the E1 algG gene Although strain E1 contains an  algG  gene that encodes theenzyme required for catalysis of the epimerization of C-5 in man-nuronate to form guluronic acid residues in AcPM, it producedpolymers without guluronic acid residues (Figs. 2 and 3). Pointmutations in  P. aeruginosa algG  have been shown to affect epimer-ization, leading to the production of alginate without  l -guluronicacid residues [14]. Because of this, we compared the E1  algG nucleotide sequence to the wild-type strain KL28. E1 containeda point mutation (G1264C) resulting in an amino acid substitu-tion(G422R)ofthewell-conservedAlgGsequencein Pseudomonas species (Fig. 4B) (Fig. S4 in Supporting Information). The mutated amino acid residue has been shown to be essential for the epimer-ization function of AlgG [28]. To confirm the proposed function of  the mutated amino acid residue, a pAlgG vector expressing wild-type KL28  algG  from KL28 was constructed and introduced into E1as described in Section 2. When the polymer formed by E1(pAlgG) wassubjectedtoacidhydrolysisandsugarcompositionanalysis,ityielded l -guluronicacidand d -mannuronicacidataratioof33–67(Fig. 2). The presence of   l -guluronic acid residues in the polymerwas also confirmed by  1 H NMR analysis (data not shown). Thisfurther defined the role of the G422 residue in the epimerizationfunction of AlgG.  3.4. Primary production of AcPM by E1 Various culture parameters for maximal AcPM production bythe E1 strain were examined. King’s B [29] Medium with 1% (w/v)ofvariouscarbonsourceswasusedfortheinitialcarbonscreening.Cultures were incubated at 30 ◦ C for 72h and the AcPM contentin the culture supernatants was measured as described in Section2. The levels of AcPM production by E1 were 0.4, 2.3, 0.2, 0.5, 0.8,0.4,3.8and0.3g/Lwithglucose,fructose,lactose,pyruvate,citrate,acetate,glycerolandsuccinate,respectively,ascarbonsources.Theresults indicated that glycerol was the best carbon source for theoptimum production of AcPM. In  P. aeruginosa  M-BR, glycerol wasalso shown to be the best carbon source for maximal alginate pro-duction [13]. AcPM production was associated with an increase in broth viscosity (1.5–42.3 ± 5.6cps) and a decrease in pH (7–5.8) of theculturemedia.AdecreaseinthepHofthemediawaspreventedby increasing the buffer strength of the King’s B Medium from 10  332  Y. Veeranagouda et al. / Process Biochemistry 46 (2011) 328–334 Fig. 5.  The effect of different concentrations of glycerol and NaCl on production of AcPM (A) and viscosity (B) by the E1 strain. Culture conditions were described inSection2.ThePMcontentandviscosityofculturebrothweremeasuredfromculture supernatants following 72h of incubation. Values represent the averages for threeindependent replicates. The standard deviation was less than 15%. to 50mM potassium phosphate. It was found that initial pH valuesof5,7,and8wereinferiortopH6.0formaximalAcPMproduction(data not shown). Based on the above results, a new productionmedium was designed in which the pH and buffer strength weremaintained at 6.0 and 50mM, respectively. In this modified King’sMedium (PG), E1 produced 4.5 ± 0.5g/L of high viscosity AcPM,and hence PG medium was used for polymer production in oursubsequent studies.In  Pseudomonas  species, increased osmolarity has been showntostimulatealginatebiosynthesis[30,31].Thus,itwasattemptedto increasetheAcPMyieldbyincorporatingNaClasanosmoticstressinducer in PG medium with varying concentrations of glycerol. Anincreaseofglycerolconcentrationinthemediafrom1%to4%(v/v)hadnosignificanteffectongrowth,AcPMproductionorAcPMvis-cosity, but 5% glycerol drastically reduced viscosity and increasedAcPM production. A large effect in the viscosity and AcPM produc-tion was also seen with the addition of NaCl to the PG medium(Fig.5AandB).Adding1%NaCltothePGmediumlinearlyincreased AcPMproductiontoalmostdouble.Interestingly,theviscositywasmaximal at 0.25% NaCl, but a dramatic reduction was observedabove 0.75%, with a viscosity nearly equal to that of water. Thisindicated that at high NaCl concentrations, the E1 strain producedlower molecular weight AcPM at an increased yield. In this study,almost8g/LofAcPMwasobtainedwiththehighestviscositybeing160cps. In addition, 14g/L of AcPM was obtained with the lowestviscosity being 1.2cps.  3.5. Analysis of the molecular weight of AcPM  The molecular weight of AcPM produced at different viscositieswas examined by gel permeation chromatography. AcPM at the Fig. 6.  Distribution of AcPM molecular weights obtained from the high (black line)and low (red line) viscosity-culture media. (For interpretation of the references tocolor in this figure legend, the reader is referred to the web version of the article.) highestviscosity(160cps)showedthreebroadpeakswithmedianmolecularweightsof415,3.4and0.95kDaataratioof4:3:3(Fig.6).This demonstrated that the high broth viscosity was mainly due tothe production of high molecular weight AcPM, though the reasonfor the distribution of these molecular weights was unclear. Fur-thermore, AcPM obtained at the lowest viscosity (1.2cps) showedhomogeneous, lower molecular weight polymers with a medianmolecular mass of 35kDa (Fig. 6).  3.6. Rheological properties of AcPM  The viscosity of an aqueous AcPM solution under different con-ditionshasnotbeenpreviouslyreported.Becausemannuronicacidisalsoacomponentofalginate,theviscosityofAcPMwascomparedto that of commercial alginate. As the polymer content increased,so did the viscosity broth of the AcPM and alginate (Fig. 7A). At a concentrationof1.5%polymer,theviscosityofAcPMwasthree-foldhigher compared to commercial alginate. The viscosities of AcPMand alginate aqueous solutions (0.25%, w/v) were determined atdifferenttemperaturesrangingfrom25to80 ◦ C(Fig.7B).Bothpoly- mershaddecreasedviscositiesathighertemperaturesbutretainedtheir srcinal viscosity when temperatures were dropped to roomtemperature. The viscosity of the AcPM solution was stable whenshifting the pH to either highly basic or moderately acidic values(Fig.7C).AtpH1.0,theviscosityofAcPMdecreasedtoabout50%;in contrast,commercialalginatelostitsviscosityandprecipitated.Theeffectsofsomerepresentativemonovalentanddivalentcationsonthe viscosity of polymer solutions were also investigated (Fig. 7D). Aslightdecreaseintheviscosityvaluesforbothpolymersolutionswas detected up to 10% (w/v) of NaCl and MgCl 2 . The determi-nation of the viscosity of commercial alginate in the presence of CaCl 2  was not possible because it formed a gel even at low CaCl 2 concentrations. In contrast, the viscosity of the AcPM solution wasnot affected even after the addition of 10% CaCl 2 . This differencewas most likely due to the presence and absence of guluronic acidresidues in polymers [11]. In fact, the commercial alginate con- tained  l -guluronic acid and  d -mannuronic acid residues at a ratioof 31:69 (Fig. 2). 4. Discussion The potential applications of PM and its derivatives in mate-rialscienceandpharmaceuticalresearchledtodevelopamicrobialsystem that efficiently produces PM. The results presented in thisstudyclearlyindicatethattheE1andE1( algI  ::Tc2)strainshavethecapacity to produce large quantities of AcPM and PM, respectively.
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