S372 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

As a result, Bjerkandera sp. L-25 was able to grow after the irra-diation over 3000 Gy of proton beam; on the contrary, it did notsurvive over 1750 Gy of carbon beam. As for the proton beam,the irradiation over 2000 Gy was effective for mutation and over1000 Gy was effective for the carbon beam. Most of the mutantsobtained by carbon beam showed a different morphology from thewild strain; on the other hand, the mutants by proton beam werenot so different from the wild strain. The mutant obtained by 3000Gy proton beam irradiation showed a higher manganese peroxidaseactivity (ca 45 U/mL) by shaking culture, which was more than 30%higher than that of the wild strain.

doi:10.1016/j.jbiotec.2010.09.446

[P-I.81]

Identification and characterization of wblA ortholog, a globalantibiotic down-regulator in Tautomycetin-producing Strepto-myces sp. CK4412

Han-Na Lee ∗, Shin-Hae Park, Si-Sun Choi, Eung-Soo Kim

Inha University, Korea, Republic ofKeywords: wblA; Tautomycetin; Streptomyces; Down-regulator

Tautomycetin (TMC), which is produced by Streptomyces sp.CK4412, is a novel activated T cell-specific immunosuppressivecompound with an ester bond linkage between a terminal cyclicanhydride moiety and a linear polyketide chain bearing an unusualterminal alkene (Choi et al., 2007). The wblA (whiB-like gene A) wasreported as a pleiotropic down-regulator of antibiotic biosynthesisof doxorubicin in S. peucetius as well as the antibiotics productionin S. coelicolor. Here, we identified a wblA ortholog gene (namedwblA-tmc) via genomic DNA library screening using the degener-ated primers based on wblA sequence from S. coelicolor A3(2) (Kanget al., 2007). A 0.39 kb wblA-tmc showed 96% amino acid identitycompared to a previously-known S. coelicolor wblA. A targeted genedisruption of wblA-tmc from the TMC-producing strain, which wasconfirmed by PCR and Southern-hybridization, exhibited signifi-cantly different morphological pattern as well as TMC productivity.In addition, complementation by an integrative plasmid carryingwblA-tmc restored TMC biosynthesis, implying that wblA-tmc isalso a key down-regulator of TMC biosynthesis. Gene expressionanalysis by RT-PCR of the TMC biosynthetic genes revealed thata wblA-tmc mutant strain exhibited reduced expression levels forthe TMC biosynthetic genes, suggesting that wblA-tmc acts globallyas a key down-regulator of TMC biosynthesis in Streptomyces sp.CK4412.

References

Choi, S.-S., Hur, Y.-A., Sherman, D.H., Kim, E.-S., 2007. Isolation of the biosyntheticgene cluster for tautomycetin, a linear polyketide T cell-specific immunomod-ulator from Streptomyces sp. CK4412. Microbiology 153, 1095–1102.

Kang, S.-H., Huang, J., Lee, H.-N., Hur, Y.-A., Cohen, S.N., Kim, E.-S., 2007. InterspeciesDNA microarray analysis identifies WblA as a pleiotropic down-regulator ofantibiotic biosynthesis in Streptomyces. J Bacteriol 189, 4315–4319.

doi:10.1016/j.jbiotec.2010.09.447

[P-I.82]

Bioproduction of polyhydroxyalkanoates by Bacillus endophyti-cus isolated from the Maharlou salt lake in south of Iran

Narges Negintaji ∗, Sara Rasoul-Amini, Younes Ghasemi

Department of Pharmaceutical Biotechnology, Faculty of Pharmacyand Pharmaceutical Sciences Research Center, Shiraz University ofMedical Sciences, Iran, Islamic Republic ofKeywords: Bioplastic,PHA; 16SrRNA; Bacillus endophyticus

Introduction: Bioplastics are natural biopolymers which aresynthesized by a variety of microorganisms and accumulated asstorage materials in microbial cells. The most widely producedmicrobial bioplastics are polyhydroxyalkanoates (PHAs) and theirderivatives. The most attractive feature of PHAs is that they canbe biodegraded into CO2 and H2O in aerobic conditions andinto methane and inorganic compounds under anaerobic condi-tions.Today there is special interest in producing PHAs to replaceconventional petrochemical plastics. The purpose of this study wasto characterize the PHA production by Bacillus endophyticus whichis isolated from the Maharlou salt lake.

Methods: The Bacillus endophyticus was isolated during a screen-ing program from the Maharlou salt lake located in the south ofIran. The bacterium bacteria was identified based upon molecularmarkers 16S rRNA by the universal prokaryotic primer. Then theisolate was stained with Sudan Black B and Nile blue A dyes to con-firm the lipidic intracellular granules and the PHA production. Thebiopolymer was extracted from the lyophilized cells and analysiedby specteroscopic methods, including UV, FT-IR and GC/MS.

Results: The Bacillus endophyticus produce PHB as the mainproduct with the mass spectrum for the methyl ester of3-hydroxybutyrate (MW = 118). The strain, which yielded anadditional spectra besides that of the methyl ester of 3-hydroxybutyrate. The mass spectra with the excellent similarityto the respective mass spectra in the MS library, confirmed thesecond peak detected in GC/MS analysis to be methyl ester of 3-hydroxyvalerate (MW = 132).

Discussion: The Bacillus endophyticus produces co-polymers ofpolyhydroxybutyrate and polyhydroxyvalerate which are stronglyremarked for industrial applications.

doi:10.1016/j.jbiotec.2010.09.448

[P-I.83]

The use of Aspergillus niger for the removal of phosphorous andpotassium from the iron ore concentrate of the Sishen Iron OreMine, South Africa

P.J. Williams 1,∗, T.E. Cloete 2

1 University of Pretoria, South Africa2 Stellenbosch University, South AfricaKeywords: Aspergillus niger; Iron ore concentrate; phosphorous;potassium

The depletion of the richer iron ore worldwide has made it nec-essary to process lower quality iron ore. Certain alkali substances,such as phosphorous (P) and potassium (K), contained within theiron ore have a detrimental effect on the smelting process duringsteel manufacturing. Therefore, international steel making com-panies charge penalties when purchasing iron ore concentratescontaining high concentrations of P and K. It has, therefore, becomenecessary to develop an economically viable and environmentallyfriendly process to reduce the high P and K concentrations con-

Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S373

tained in the iron ore concentrate of the Sishen Iron Ore Mine.Conventional bioleaching refers to the microbial conversion of

insoluble metals into soluble forms, however, the P and K con-tained within the iron ore concentrate are in non-sulphidic phases,and therefore, conventional bioleaching processes are not viablefor their removal from the ore. Therefore, the use of an alternativebioleaching microorganism has to be investigated to remove theundesirable P and K from the iron ore concentrate. The objective ofthis study is, therefore, to investigate the possible use of A. niger ina bioleaching process for the economic removal of P and K from theiron ore concentrate of the Sishen Iron Ore Mine.

During this study different pulp densities [bioleaching solution(ml)/iron ore (g)] were compared for the removal of P and K fromthe iron ore concentrate using A. niger as the bioleaching microor-ganism. A pulp density of 33% proved to be most efficient in terms ofcitric acid production, producing a maximum of 52.06 g.l-1 of citricacid at 30 ◦C after 96 h. In addition, the 33% pulp density resulted inthe maximum P and K removal of 23.53% and 17.65% respectively,and therefore, A. niger can be considered as a suitable bioleachingcandidate for the removal of P and K from the iron ore concentrateof the Sishen Iron Ore Mine.

doi:10.1016/j.jbiotec.2010.09.449

[P-I.84]

Industrial Production of threo-3-Hydroxy-L-Aspartic Acid UsingEscherichia coli Resting Cells

R. Hara ∗, K. Kino

Department of Applied Chemistry, Faculty of Science and Engineering,Waseda University, JapanKeywords: threo-3-hydroxy-L-aspartic acid; hydroxyaspartic acid

In nature, 3-hydorxyaspartic acid is found as free form oras peptide constituent in various microbial and fungal metabo-lites. This non-proteinous amino acid contains two chiral centersand thus four stereoisomers, those are threo-3-hydroxy-L-asparticacid (L-THA), erythro-3-hydroxy-L-aspartic acid, threo-3-hydroxy-D-aspartic acid, and erythro-3-hydroxy-D-aspartic acid. L-THA is avery useful compound for a pharmaceutical, because L-THA itselfshows potent inhibitory activity against various strains of humanpathogenic fungi. But, regio- and stereo-selective synthesis of L-THA is difficult by a conventional chemical synthesis. Here weestablished the industrial process of L-THA production using aminoacid hydroxylase.

Recently, the tertiary structure of L-asparagine hydroxylase(AsnO) from Streptomyces coelicolor A3(2), which can hydroxylateC-3 position of L-asparagine, is elucidated (Strieker et al., 2007).Furthermore, the AsnO mutant, which is AsnO-D241N, showedthe hydroxylation of L-aspartic acid (Strieker et al., 2008). Wedeveloped the production process of L-THA using resting cellsof Escherichia coli containing AsnO-D241N. L-THA was directlyproduced from L-aspartic acid as a substrate; however, the conver-sion yield was not sufficient due to low activity and instability ofAsnO-D241N. We confirmed the deamidation of threo-3-hydorxy-L-aspargine by asparaginase (EC 3.5.1.1). Then, we contrived theAsnO-asparaginase coupling reaction for L-THA production fromL-asparagine. When E. coli expressing asnO was used, L-THA wasproduced in good yield from L-asparagine as a substrate. This resultsuggested that L-asparagine was hydroxylated by L-asparaginehydroxylase and subsequently the product, threo-3-hydroxy-L-asparagine, was converted to L-THA by asparaginase of E. coli nativeenzyme.

References

Strieker, M., Kopp, F., Mahlert, C., Essen, L.O., Marahiel, M.A., 2007. ACS Chem. Biol.2, 187–196.

Strieker, M., Essen, L.O., Walsh, C.T., Marahiel, M.A., 2008. Chembiochem 9, 374–376.

doi:10.1016/j.jbiotec.2010.09.450

[P-I.85]

Evolutionary engineering of Actinobacillus succinogenes forimproved succinic acid production on glycerol

Christophe Roca ∗, Graca Albuquerque, Maria Reis

FCT/Universidade Nova de Lisboa, PortugalKeywords: succinic acid; glycerol; Actinobacillus succinogenes

Succinic acid has attracted much attention as building blockused in a variety of commodity chemicals, ranging from fibers tosolvents and pharmaceuticals. However, its high production cost,both chemically from petroleum-based maleic anhydride and bio-logically from fermentation of various carbon sources, preventedits industrial development. Efficient fermentation of cheap andrenewable feedstocks has been proposed as a way to turn thefermentation process economically viable and cost competitivewith the petroleum-based succinic acid. Therefore, new feedstocks,besides agricultural waste residues or lignocellulosic materials,have to be tested, such as industrial effluents. Glycerol has recentlyappeared as a new cheap feedstock, largely available as a by-product from the biodiesel industry. Actinobacillus succinogeneshas been isolated from bovine rumen and is one of the bestproducers of succinic acid, together with Mannheimia succinopro-ducens or Anaerobiospririllum succiniciproducens (McKinley et al.,2005). However, conversion of glycerol by this bacterium remainsslow and incomplete, requiring dramatic improvement to becomeindustrially efficient. For example, continuous cultivations withBasfia succiniproducens, closely related to A. succinogenes, could notexceed a dilution rate of 0.02 h-1 on crude glycerol, before substrateaccumulation (Schoten et al., 2009). Evolutionary engineering hasbeen successfully applied to obtain improved phenotypes of indus-trially relevant microorganisms such as Saccharomyces cerevisiae orEscherichia coli (Sauer, 2001). In the present work, we will designevolutionary engineering strategies to improve glycerol utilizationby A. succinogenes. Repetitive batch cultures and continuous culturewill be performed in order to obtain an improved strain populationadapted to glycerol, able to maintain high succinic acid produc-tion yield and productivity. Adapted/mutated strain will be furtheranalyzed to identify the cellular modifications responsible for phe-notype improvement.

References

McKinley, et al., 2005. Applied and Environmental Microbiology 71 (11), 6651–6656.Schoten, et al., 2009. Biotechnology Letters 31 (12), 1947–1951.Sauer, U., 2001. Adv. Biochemical Engineering/Biotechnology 73, 129–169.

doi:10.1016/j.jbiotec.2010.09.451