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Applied Microbiology and Biotechnology (v.72, #5)

Lactic acid production from xylose by the fungus Rhizopus oryzae by Ronald H. W. Maas; Robert R. Bakker; Gerrit Eggink; Ruud A. Weusthuis (pp. 861-868).

Lignocellulosic biomass is considered nowadays to be an economically attractive carbohydrate feedstock for large-scale fermentation of bulk chemicals such as lactic acid. The filamentous fungus Rhizopus oryzae is able to grow in mineral medium with glucose as sole carbon source and to produce optically pure l(+)-lactic acid. Less is known about the conversion by R. oryzae of pentose sugars such as xylose, which is abundantly present in lignocellulosic hydrolysates. This paper describes the conversion of xylose in synthetic media into lactic acid by ten R. oryzae strains resulting in yields between 0.41 and 0.71 g g⁻¹. By-products were fungal biomass, xylitol, glycerol, ethanol and carbon dioxide. The growth of R. oryzae CBS 112.07 in media with initial xylose concentrations above 40 g l⁻¹ showed inhibition of substrate consumption and lactic acid production rates. In case of mixed substrates, diauxic growth was observed where consumption of glucose and xylose occurred subsequently. Sugar consumption rate and lactic acid production rate were significantly higher during glucose consumption phase compared to xylose consumption phase. Available xylose (10.3 g l⁻¹) and glucose (19.2 g l⁻¹) present in a mild-temperature alkaline treated wheat straw hydrolysate was converted subsequently by R. oryzae with rates of 2.2 g glucose l⁻¹ h⁻¹ and 0.5 g xylose l⁻¹ h⁻¹. This resulted mainly into the product lactic acid (6.8 g l⁻¹) and ethanol (5.7 g l⁻¹).

Production of lipopeptide antibiotic iturin A using soybean curd residue cultivated with Bacillus subtilis in solid-state fermentation by S. Mizumoto; M. Hirai; M. Shoda (pp. 869-875).

Bacillus subtilis RB14-CS, which suppresses the growth of various plant pathogens in vitro by producing the lipopeptide antibiotic iturin A, was cultured using soybean curd residue, okara, a by-product of tofu manufacture in solid-state fermentation. After 4 days incubation, iturin A production reached 3,300 mg/kg wet solid material (14 g/kg dry solid material), which is approximately tenfold higher than that in submerged fermentation. When the okara product cultured with RB14-CS was introduced into soil infested with Rhizoctonia solani, which is a causal agent of damping-off of tomato, the disease occurrence was significantly suppressed. After 14 days, the number of RB14-CS cells remained in soil at the initial level, whereas almost no iturin A was detected in soil. As the okara cultured with RB14-CS exhibited functions of both plant disease suppression and nutritional effect on tomato seedlings, this product is expected to contribute to the recycling of the soybean curd residue.

Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319 by Luo Liu; Rolf D. Schmid; Vlada B. Urlacher (pp. 876-882).

A new member of class IV of cytochrome P450 monooxygenases was identified in Rhodococcus ruber strain DSM 44319. As the genome of R. ruber has not been sequenced, a P450-like gene fragment was amplified using degenerated primers. The flanking regions of the P450-like DNA fragment were identified by directional genome walking using polymerase chain reaction. The primary protein structure suggests a natural self-sufficient fusion protein consisting of ferredoxin, flavin-containing reductase, and P450 monooxygenase. The only flavin found within the enzyme was riboflavin 5′-monophosphate. The enzyme was successfully expressed in Escherichia coli, purified and characterized. In the presence of NADPH, the P450 monooxygenase showed hydroxylation activity towards polycyclic aromatic hydrocarbons naphthalene, indene, acenaphthene, toluene, fluorene, m-xylene, and ethyl benzene. The conversion of naphthalene, acenaphthene, and fluorene resulted in respective ring monohydroxylated metabolites. Alkyl aromatics like toluene, m-xylene, and ethyl benzene were hydroxylated exclusively at the side chains. The new enzyme’s ability to oxidize such compounds makes it a potential candidate for biodegradation of pollutants and an attractive biocatalyst for synthesis.

Biotechnologically relevant enzymes and proteins by Ana Beatriz Moreno; Álvaro Martínez del Pozo; Blanca San Segundo (pp. 883-895).

The mold Aspergillus giganteus produces a basic, low molecular weight protein showing antifungal properties against economically important plant pathogens, the AFP (Antifungal Protein). In this study, we investigated the mechanisms by which AFP exerts its antifungal activity against Magnaporthe grisea. M. grisea is the causal agent of rice blast, one of the most devastating diseases of cultivated rice worldwide. AFP was purified from the extracellular medium of A. giganteus cultures. The AFP protein was found to induce membrane permeabilization in M. grisea cells. Electron microscopy studies revealed severe cellular degradation and damage of plasma membranes in AFP-treated fungal cells. AFP however failed to induce membrane permeabilization on rice or human HeLa cells. Furthermore, AFP enters the fungal cell and targets to the nucleus, as revealed by co-localization experiments of Alexa-labeled AFP with the SYTOX Green dye. Finally, AFP binds to nucleic acids, including M. grisea DNA. Our results suggest that the combination of fungal cell permeabilization, cell-penetrating ability and nucleic acid-binding activity of AFP determines its potent antifungal activity against M. grisea. These results are discussed in relation to the potential of the AFP protein to enhance crop protection against fungal diseases.

Mutation on N-terminus of polyhydroxybutyrate synthase of Ralstonia eutropha enhanced PHB accumulation by Zhong Zheng; Ming Li; Xiao-Jing Xue; Hong-Lei Tian; Zhi Li; Guo-Qiang Chen (pp. 896-905).

Polyhydroxyalkanoate (PHA) synthase is the central enzyme involved in the biosynthesis of PHA, a family of bacterial biodegradable polyesters. Due to its high variability, the N-terminal fragment of this enzyme was previously considered as unnecessary for a functionally active enzyme. In this study, polyhydroxybutyrate synthase from Ralstonia eutropha (PhbC_Re) with a deletion on N-terminal 88 amino acid residues showed a significant reduced activity, as reflected by only 1.5% PHB accumulation compared with the wild type which produced 58.4% PHB of the cell dry weight. Whilst several site-specific mutagenesis results revealed the amphiphilic α-helix assembled by the amino acid region, D70–E88 played an important role in both maintaining the PHB synthase activity and regulating molecular weight and polydispersity of accumulated PHB homopolymer.

Functional characterization of alcohol oxidases from Aspergillus terreus MTCC 6324 by A. Kiran Kumar; Pranab Goswami (pp. 906-911).

Short chain alcohol oxidase (SCAO), long chain alcohol oxidase (LCAO), secondary alcohol oxidase (SAO), and aryl alcohol oxidase (AAO) activities were localized in the microsome of Aspergillus terreus during growth of the fungi on n-hexadecane. Zymogram analysis of the microsomes of n-hexadecane-grown cells in polyacrylamide gel electrophoresis showed distinct bands, H4, H3, H2, and H1, in a sequence of their molecular weight (Mr) from high to low. The Mr of the isozymes corresponding to the bands H4, H3, and H2 were close to each other and were higher than 272 kDa. While, the Mr of the isozyme H1 was found to be approximately 48 kDa. H1 gave activity only as SCAO. Although the substrates for other bands were varied, strong (S), medium (M), and weak (W) activity for the bands were as follows: H2: SAO (S), AAO (S), LCAO (M), SCAO (S); H3: LCAO (S), SCAO (S); H4: SCAO (S), LCAO (W), SAO (W). The pH and temperature optima of these isozymes were found to be 8.5±0.5 and 30±1°C, respectively. The stability of the isozymes was drastically decreased beyond 30°C. The SAO showed 33% enantiomeric excess for the R(−)2-octanol over S(+)2-octanol, which may be correlated with the lower Michaelis–Menten constant (K _M) values of the enzyme for the R(−)2-octanol than the S(+)2-octanol. The fluorescence emission spectra of the chromatographically purified SCAO at 443 nm excitation were similar to that obtained with authentic flavin adenine dinucleotide.

A ribonuclease from the wild mushroom Boletus griseus by Hexiang Wang; T. B. Ng (pp. 912-916).

A ribonuclease (RNase) with a molecular mass of 29 kDa and cospecific for poly A and poly U was isolated from fruiting bodies of the mushroom Boletus griseus. Its N-terminal sequence exhibited some similarity to those of RNases from the mushrooms Irpex lacteus and Lentinus edodes. The RNase was adsorbed on diethylaminoethyl-cellulose, Q-Sepharose, and Affi-gel blue gel and was unadsorbed on CM-cellulose. The enzyme exhibited a temperature optimum between 60 and 70°C and a pH optimum at 3.5.

TspMI, a thermostable isoschizomer of XmaI (5′C/CCGGG3′): characterization and single molecule imaging with DNA by Vijay Parashar; Neena Capalash; Shuang-yong Xu; Yasushi Sako; Prince Sharma (pp. 917-923).

TspMI, a thermostable isoschizomer of XmaI from a Thermus sp., has been characterized. The enzyme was purified to homogeneity using Cibacron-Blue 3GA agarose, Heparin agarose, SP sephadex C50, and Mono-Q fast protein liquid chromatography and was found to be a homodimer of 40 kDa. Restriction mapping and run-off sequencing of TspMI-cleaved DNA ends depicted that it cleaved at 5′C/CCGGG3′ to generate a four-base, 5′-CCGG overhang. The enzyme was sensitive to methylation of second and third cytosines in its recognition sequence. TspMI worked optimally at 60°C with 6 mM Mg²⁺, no Na⁺/K⁺, and showed no star activity in the presence of 25% glycerol. The enzyme could efficiently digest the DNA labeled with a higher concentration of YOYO-I (one dye molecule to one nucleotide), making it a useful candidate for real-time imaging experiments. Single molecule interaction between TspMI and λ DNA was studied using total internal reflection fluorescence microscopy. The enzyme survived 30 polymerase chain reaction (PCR) cycles in the presence of 10% glycerol and 0.5 M trehalose without any activity loss and, hence, is suitable for incorporation in restriction-endonuclease-mediated selective-PCR for various applications.

Engineered Bacillus thuringiensis GO33A with broad insecticidal activity against lepidopteran and coleopteran pests by Guangjun Wang; Jie Zhang; Fuping Song; Jun Wu; Shuliang Feng; Dafang Huang (pp. 924-930).

A recombinant plasmid pSTK-3A containing cry3Aa7 gene encoding a coleopteran-specific insecticidal protein was constructed and introduced into wild Bacillus thuringiensis subsp. aizawai G03, which contained cry1Aa, cry1Ac, cry1Ca, and cry2Ab genes and was highly toxic to lepidopteran insect pests. The genetically engineered strain were named G033A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis demonstrated that the cry3Aa7 gene was expressed normally and produced a 67 kDa protein in G033A, and the flat rectangular crystals of Cry3Aa7 toxin protein was observed under scanning electron microscope. The recombinant plasmid was maintained in bacteria cultured for 180 generations in culture media containing no antibiotics. Synthesis of the Cry3Aa7 toxin conferred high and broad toxicity to the recombinant strain G033A against coleopteran order, elm leaf beetle (Pyrrhalta aenescens) (LC₅₀ 0.35 mg/ml), for which the parental strain G03 was not toxic. Both the parental strain G03 and recombinant strain G033A showed strong insecticidal activity to lepidopteran pests, beet armyworm (Spodoptera exigua), diamondback moth (Plutella xylostella), and cotton bollworm (Helicoverpa amigera), respectively. The lethal concentration 50% (LC₅₀) of G033A against S. exigua, P. xylostella, and H. amigera was 4.26, 0.86, and 1.76 μg/ml, respectively.

High yield expression of Lipase A from Candida antarctica in the methylotrophic yeast Pichia pastoris and its purification and characterisation by Jan Pfeffer; Sven Richter; Jens Nieveler; Carl-Erik Hansen; Rachid Bel Rhlid; Rolf D. Schmid; Monika Rusnak (pp. 931-938).

The current investigation focuses on shedding further light on the characteristics of lipase A from Candida antarctica (CalA), which has attracted growing attention in its suitability for industrial applications. CalA was functionally expressed in the methylotrophic yeast Pichia pastoris, purified and characterised. A classical fed-batch process and a semi-continuous process were developed and tested with regard to their yield capacity. Lipase concentrations of 0.88 and 0.55 g l⁻¹ were obtained using the fed-batch and semi-continuous processes, respectively. The semi-continuous process reaches a total activity of 10,233,000 U and so surpasses the fed-batch process reaching 7,530,000 U. The purified enzyme showed highest activity between 50 and 70 °C at pH 7.0 and a preference for short-chain triglycerides (C4-C8). Significantly reduced activity was observed in the presence of hydrophilic esters.

Purification and characterization of laccase produced by a white rot fungus Pleurotus sajor-caju under submerged culture condition and its potential in decolorization of azo dyes by Kumarasamy Murugesan; Manavalan Arulmani; In-Hyun Nam; Young-Mo Kim; Yoon-Seok Chang; P. Thangavelu Kalaichelvan (pp. 939-946).

An extracellular laccase was isolated and purified from Pleurotus sajor-caju grown in submerged culture in a bioreactor, and used to investigate its ability to decolorize three azo dyes. The extracellular laccase production was enhanced up to 2.5-fold in the medium amended with xylidine (1 mM). Purification was carried out using ammonium sulfate (70% w/v), DEAE-cellulose, and Sephadex G-100 column chromatography. The enzyme was purified up to 10.3-fold from the initial protein preparation with an overall yield of 53%. The purified laccase was monomeric with an apparent molecular mass of 61.0 kDa. The purified enzyme exerted its optimal activity with 2,2-azino–bis(3-ethylbenzo-thiazoline-6-sulfonate (ABTS) and oxidized various lignin-related phenols. The catalytic efficiencies k _cat/K _m determined for ABTS and syringaldazine were 9.2×10⁵ and 8.7×10⁵, respectively. The optimum pH and temperature for the purified enzyme was 5.0 and 40 °C, respectively. Sodium azide completely inhibited the laccase activity. The absorption spectrum revealed type 1 and type 3 copper signals. The purified enzyme decolorized azo dyes such as acid red 18, acid Black 1, and direct blue 71 up to 90, 87, and 72%, respectively. Decolorization ability of P. sajor-caju laccase suggests that this enzyme could be used for decolorization of industrial effluents.

Chromosome-shuffling technique for selected chromosomal segments in Saccharomyces cerevisiae by Minetaka Sugiyama; Eishi Yamamoto; Yukio Mukai; Yoshinobu Kaneko; Masafumi Nishizawa; Satoshi Harashima (pp. 947-952).

We describe a novel chromosome engineering technique for shuffling selected regions of chromosomes from two strains in Saccharomyces cerevisiae: The technique starts with the construction of MAT a and MATα strains in which a particular chromosome is split at exactly the same site in both strains such that the split chromosomes generated are marked with different markers. The two strains are then crossed, and the resultant diploid is cultivated in nutrient medium to induce loss of the split chromosome originating from either of the strains. We predicted that some of these clones that are hemizygous for the split chromosome would spontaneously restore a homozygous configuration of the split chromosome during cultivation. We verified this prediction by tetrad analysis and quantitative Southern hybridization analysis, indicating that it is possible to create diploid hybrids in which a selected region of a chromosome from one strain is replaced by the corresponding chromosomal region from another strain. We also found that some chromosomal segments maintain a hemizygous state. This novel technique, which we call ‘chromosome shuffling’, could provide a new tool to analyze phenotypic alterations caused by the replacement or hemizygosity of a selected chromosomal region in not only laboratory but also industrial strains of S. cerevisiae.

Enhancing enzymatic activity of penicillin G acylase by coexpressing pcm gene by Tianwen Wang; Hu Zhu; Xingyuan Ma; Zhuoya Fei; Yushu Ma; Dongzhi Wei (pp. 953-958).

Penicillin G acylase (PGA; E.C. 3.5.1.11) is an important enzyme which has broad applications in industries of β-lactim antibiotics production. In this study, a promising PGA gene from Alcaligenes faecalis (afpga) and another pcm gene encoding protein isoaspartate methyltransferase (PIMT) were constructed into pET43.1a⁽⁺⁾ and pET28a⁽⁺⁾, respectively. The recombinant plasmids pETAFPGA and pETPCM were transformed into the same host cell Escherichia coli BL21 (DE3). Results suggested that the two plasmids could peacefully exist in the host cell and the two genes could be efficiently expressed after induction. The product of pcm gene could function as a helper molecule for enzyme AFPGA. PIMT increased the enzymatic activities in supernatant of ferment broth (1.6 folds) and cell lysate (1.8 folds), while it did not significantly affect the expression level of penicillin G acylase.

Protein splicing of PRP8 mini-inteins from species of the genus Penicillium by Skander Elleuche; Nicole Nolting; Stefanie Pöggeler (pp. 959-967).

Inteins are protein-intervening sequences found inside the coding region of different host proteins and are translated in-frame with them. They can self-excise through protein splicing, which ligates the host protein flanks with a peptide bond. In this study, four different species of the genus Penicillium were investigated for the presence of inteins inside the conserved splicing-factor protein PRP8. We identified 157 to 162 amino acid in-frame insertions in the PRP8 protein of Penicillium chrysogenum, Penicillium expansum, and Penicillium vulpinum (formerly Penicillium claviforme). The Penicillium PRP8 inteins are mini-inteins without a conserved endonuclease domain. We demonstrated that the PRP8 mini-inteins of P. chrysogenum, P. expansum, and P. vulpinum undergo autocatalytic protein splicing when heterologously expressed in E. coli, in a model host protein, and in a divided GFP model system. They are, thus, among the smallest known nuclear-encoded, active splicing protein elements. The GFP assay should be valuable as a screening system for protein splicing inhibitors as potential antimycotic agents and as tools for studying the mechanism of protein splicing of fungal mini-inteins.

Characterization of lycopene-overproducing E. coli strains in high cell density fermentations by Hal Alper; Kohei Miyaoku; Gregory Stephanopoulos (pp. 968-974).

Previous work identified two recombinant strains of Escherichia coli capable of significant lycopene overproduction. These strains were constructed by superimposing the deletion of three genes, selected through combinatorial and systematic searches of the metabolic landscape, onto a previously engineered strain over-expressing critical genes in the lycopene biosynthesis pathway. In this paper, we characterize the performance of these two strains in comparison to the parental, pre-engineered strain. Specifically, high cell density fermentations were performed after identifying optimized putative operating parameters. High oxygen levels and increased pH values were found to be critical for increasing both specific and volumetric product titers. Carbon balances suggest linkages between glutamate, NADPH, formate, and alanine levels with lycopene overproduction. Furthermore, lycopene production reached nearly 220 mg/l from approximately 27 g dry cell weight/l in these reactors, which is the highest value reported to date for E. coli.

A functional analysis of the Bifidobacterium longum cscA and scrP genes in sucrose utilization by B. Kullin; V. R. Abratt; S. J. Reid (pp. 975-981).

The role of genes involved in sucrose catabolism was investigated with a view to designing effective prebiotic substrates to encourage the growth of Bifidobacterium in the gut. Two gene clusters coding for sucrose utilisation in Bifidobacterium longum NCC2705 were identified in the published genome. The genes encoding putative sucrose degrading enzymes, namely, the scrP (sucrose phosphorylase) and the cscA (β-fructofuranosidase), were cloned from B. longum NCIMB 702259^T and expressed in Escherichia coli DH5α. Both complemented the sucrase negative phenotype of untransformed cells and showed specific sucrase activity. Transcriptional analysis of the expression of the genes in B. longum grown in the presence of various carbohydrate substrates showed induction of scrP gene expression in the presence of sucrose and raffinose, but not in the presence of glucose. The cscA gene showed no increased transcription in B. longum grown in the presence of any of the carbohydrates tested. Phylogenetic analysis indicates that the B. longum CscA protein belongs to a distinct phylogenetic cluster of intracellular fructosidases, which specifically cleave the shorter fructose oligosaccharides.

Amplification of 1-deoxy-d-xyluose 5-phosphate (DXP) synthase level increases coenzyme Q₁₀ production in recombinant Escherichia coli by Soo-Jung Kim; Myoung-Dong Kim; Jin-Ho Choi; Sang-Yong Kim; Yeon-Woo Ryu; Jin-Ho Seo (pp. 982-985).

For the enhancement of coenzyme Q₁₀ (CoQ₁₀) production, 1-deoxy-d-xylulose 5-phosphate (DXP) synthase of Pseudomonas aeruginosa was constitutively coexpressed in a recombinant Escherichia coli strain, which harbors the ddsA gene from Gluconobacter suboxydans encoding decaprenyl diphosphate synthase. It was found that the expression of the ddsA gene caused depletion of the isopentenyl diphosphate (IPP) pool in E. coli. Amplification of DXP synthase level by installing P. aeruginosa DXP synthase restored the diminished IPP pool and concomitantly resulted in approximately a twofold increase in relative content and productivity of CoQ₁₀. Maximum CoQ₁₀ concentration of 46.1 mg l⁻¹ was achieved from glucose-limited fed-batch cultivation of the recombinant E. coli strain simultaneously harboring the ddsA and dxs genes.

Construction of ivermectin producer by domain swaps of avermectin polyketide synthase in Streptomyces avermitilis by Xiaolin Zhang; Zhi Chen; Meng Li; Ying Wen; Yuan Song; Jilun Li (pp. 986-994).

Ivermectin, 22, 23-dihydroavermectin B1, is commercially important in human, veterinary medicine, and pesticides. It is currently synthesized by chemical reduction of the double bond between C22 and C23 of avermectins B1, which are a mixture of B1a (>80%) and B1b (<20%) produced by fermentation of Streptomyces avermitilis. The cost of ivermectin is much higher than that of avermectins B1 owing to the necessity of region-specific hydrogenation at C22–C23 of avermectins B1 with rhodium chloride as the catalyst for producing ivermectin. Here we report that ivermectin can be produced directly by fermentation of recombinant strains constructed through targeted genetic engineering of the avermectin polyketide synthase (PKS) in S. avermitilis Olm73-12, which produces only avermectins B and not avermectins A and oligomycin. The DNA region encoding the dehydratase (DH) and ketoreductase (KR) domains of module 2 from the avermectin PKS in S. avermitilis Olm73-12 was replaced by the DNA fragment encoding the DH, enoylreductase, and KR domains from module 4 of the pikromycin PKS of Streptomyces venezuelae ATCC 15439 using a gene replacement vector pXL211. Twenty-seven of mutants were found to produce a small amount of 22, 23-dihydroavermectin B1a and avermectin B1a and B2a by high performance liquid chromatography and liquid chromatography mass spectrometry analysis. This study might provide a route to the low-cost production of ivermectin by fermentation.

Cloning, functional expression and promoter analysis of xylanase III gene from Trichoderma reesei by W. Ogasawara; Y. Shida; T. Furukawa; R. Shimada; S. Nakagawa; M. Kawamura; T. Yagyu; A. Kosuge; J. Xu; M. Nogawa; H. Okada; Y. Morikawa (pp. 995-1003).

In this study, the xyn3 gene from the filamentous mesophilic fungus Trichoderma reesei (Hypocrea jecorina) PC-3-7 was cloned and sequenced. Analysis of the deduced amino acid sequence of XYN III revealed considerable homology with xylanases belonging to glycoside hydrolase family 10. These results show that XYN III is distinguishable from XYN I and XYN II, two other T. reesei xylanases that belong to the glycosidase family 11. When xyn3 was expressed in Escherichia coli, significant activity was observed in the cell-free extract, and higher activity (13.2 U/ml medium) was recovered from the inclusion bodies in the cell debris. The sequence of the 5′-upstream region of the gene in the parent strain QM9414 is identical to that of PC-3-7, although the expression level of xyn3 in PC-3-7 has been reported to be at least 1,000 times greater than in QM9414. These results suggest that xyn3 expression in T. reesei QM9414 is silenced. The consensus sequences for ACEI, ACEII, CREI, and the Hap2/3/5 protein complex are all present in the upstream region of xyn3. Deletion analysis of the upstream region revealed that two regions containing consensus sequences for the known regulatory elements play important roles for xyn3 expression.

Development and application of real-time PCR for quantification of specific ammonia-oxidizing bacteria in activated sludge of sewage treatment systems by Tawan Limpiyakorn; Futoshi Kurisu; Osami Yagi (pp. 1004-1013).

In this study, four real-time polymerase chain reaction (PCR) primer sets were developed for the 16S rRNA genes of specific ammonia-oxidizing bacteria (AOB) found in activated sludge of sewage treatment systems. The primer sets target two of several sequence types of the Nitrosomonas oligotropha cluster, members within the Nitrosomonas communis cluster, and all members of the Nitrosomonas europaea–Nitrosococcus mobilis cluster. The detection limit of each primer set was in the range of 3×10¹–6×10² genes reaction⁻¹. Reliable quantification of the target AOB DNA was obtained when the target AOB DNA comprised more than 0.1% of total AOB DNA in the sample. The application of the primer sets to samples taken from five sewage treatment systems showed that, in all systems, the majority of the AOB population was comprised of one sequence type of the N. oligotropha cluster (3.9±1.5×10⁹–1.7±0.5×10¹⁰ cell l⁻¹) and, in most systems, followed by members within the N. communis cluster (2.8±0.3×10⁹–1.0±0.1×10¹⁰ cell l⁻¹) or/and another sequence type of the N. oligotropha cluster (1.5±0.6×10⁸–5.5±0.5×10⁸ cell l⁻¹). N. europaea–N. mobilis cluster arose solely in small numbers (4.9±0.8×10⁸ cell l⁻¹) in one system. Real-time PCR-amplified products obtained from genomic DNA extracted from samples were verified using clone library, and it revealed that only the target AOB DNA were PCR amplified, without amplification of the nontarget sequences.

Marker and promoter effects on heterologous expression in Aspergillus nidulans by David Lubertozzi; Jay D. Keasling (pp. 1014-1023).

To study the effects of selection marker, promoter type, and copy number on heterologous expression in Aspergillus nidulans, strains were constructed with single- and multicopy plasmid integrations bearing a reporter gene (lacZ) under the control of either an inducible (alcA) or constitutive (gpdA) promoter and one of three Aspergillus nutritional marker genes (argB, trpC, or niaD). β-Galactosidase activity in the transformants varied over three orders of magnitude, with the majority of levels in the range of 5×10³–1×10⁴ U/mg. Significant differences in mean expression levels were found when comparing single-copy transformants with the same promoter but a different marker. Transformants with the argB marker had the highest average expression, ∼threefold over the trpC or niaD clones. For each promoter, maximal expression for the set was seen in the range of the single-copy clones, implying that increasing the copy number does not reliably increase expression in Aspergillus.

Functional expression of Candida antarctica lipase B in the Escherichia coli cytoplasm—a screening system for a frequently used biocatalyst by D. Liu; R. D. Schmid; M. Rusnak (pp. 1024-1032).

In this paper, we report for the first time the functional expression of lipase B from the yeast Candida antarctica (CalB) in the Escherichia coli cytoplasm. The enzyme possessing three disulfide bonds was functionally expressed in the strain Origami B. Expression under the control of a lac promoter yielded 2 U mg⁻¹, whereas expression of a thioredoxin–CalB fusion protein yielded 17 U mg⁻¹. The native enzyme was most efficiently expressed under control of the cspA promoter (11 U mg⁻¹). Coexpression of different chaperones led to a strong increase in active protein formation (up to 61 U mg⁻¹). A codon-optimized synthetic variant of calb did not show significant effects on functional protein yield. Functional CalB expression was not only achieved in shake flasks but also in microtiter plate scale. Therefore, this CalB expression system is suitable for high-throughput applications, including the screening of large gene libraries as those derived from directed evolution experiments.

Functional solubilization of aggregation-prone TRAIL protein facilitated by coexpressing with protein isoaspartate methyltranferase by Hu Zhu; Ruo-Jun Pan; Tian-Wen Wang; Ya-Ling Shen; Dong-Zhi Wei (pp. 1033-1038).

TRAIL was a tumor-specific protein in development as a novel anticancer therapeutic agent. Generally, when expressed in recombinant Escherichia coli, TRAIL protein was prone to form inclusion bodies. In this study, coexpression of human TRAIL protein and protein isoaspartate methyltranferase (PIMT) from E. coli on plasmid pBV–TRAIL–PCM in E. coli C600 was investigated to overcome the difficulties in soluble expression. The results showed that this PIMT coexpression strategy exerted a positive effect on the TRAIL protein expression in recombinant E. coli, which led to a mean increase in the intracellular concentration of soluble and total protein of TRAIL by 1.57-fold and 1.33-fold, respectively. At the same time, results also suggested that PIMT was a prospective partner for soluble expression of TRAIL protein.

High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris by Ai-Sheng Xiong; Quan-Hong Yao; Ri-He Peng; Zhen Zhang; Fang Xu; Jin-Ge Liu; Pei-Lai Han; Jian-Min Chen (pp. 1039-1047).

Phytase is widespread in nature. It has been used as a cereal feed additive that can enhance the phosphorus and mineral absorption in monogastric animals to reduce the level of phosphorus output in manure. Phytase of Peniophora lycii is a 6′-phytase, which owns high specific activity. To achieve a high expression level of 6′-phytase in Pichia pastoris, the 1,230-bp phytase gene of P. lycii was synthesized and optimized for codon usage, G+C content, as well as mRNA secondary structures. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene (AOX1) promoter, the synthetic signal peptide (designated MF4I), which is a codon-modified Saccharomyces cerevisiae mating factor α-prepro-leader sequence, were used to transform P. pastoris. The P. pastoris strain that expressed the modified phytase gene (phy-pl-sh) with MF4I sequence produced 12.2 g phytase per liter of fluid culture, with the phytase activity of 10,540 U ml⁻¹. The yield of the modified phytase gene, with bias codon usage and MF4I signal, is 4.4 times higher than that of the wild type gene with MF4I signal and 13.6 times higher than that of the wild type gene with wild type S. cerevisiae signal. The recombinant phytase had one optimum pH (pH 4.5) and an optimum temperature of 50°C. The P. pastoris strain expressed the modified 6-phytase gene, with the MF4I signal peptide showing great potential as a commercial phytase production system.

Deletion analysis of the superoxide dismutase (sodM) promoter from Aspergillus oryzae by Hiromoto Hisada; Motoaki Sano; Hiroki Ishida; Yoji Hata; Yasuhisa Abe; Masayuki Machida (pp. 1048-1053).

The manganese superoxide dismutase gene (sodM) is very highly expressed in Aspergillus oryzae. To elucidate the basis for this high-level expression, deletion analysis of the promoter was undertaken using β-glucuronidase (GUS) as a reporter. Deletion of a 63-bp sequence from −200 to −138 in the 1,038-bp sodM promoter caused a drastic decrease in GUS activity. In addition, an electrophoretic gel mobility shift assay (EMSA) implicated a 30-bp element from −209 to −178 containing cis-element(s) in the high-level expression. The results of fine structure deletion analysis of this region were consistent with the EMSA results. To confirm these findings, we constructed enhanced sodM promoters by incorporating tandem repeats of this region, which resulted in an approximate twofold increase in expression relative to the native sodM promoter.

Regulation of polyhydroxyalkanoate synthases (phaC1 and phaC2) gene expression in Pseudomonas corrugata by Enrico Conte; Vittoria Catara; Sebastiana Greco; Marcella Russo; Rossana Alicata; Luciana Strano; Alessandro Lombardo; Silvia Di Silvestro; Antonino Catara (pp. 1054-1062).

In this study we examined polyhydroxyalkanoate (PHA) synthases phaC1 and phaC2 gene expression in two strains of Pseudomonas corrugata (Pc) grown in a minimum mineral medium with related (oleic acid and octanoate) or unrelated (glucose) carbon sources. Analysis of transcription was performed by Northern blot and conventional reverse transcriptase (RT) polymerase chain reaction (PCR). In addition, we developed a RT-real-time PCR method to quantitatively evaluate phaC1 _Pc and phaC2 _Pc gene expression. Primers and a TaqMan probe were designed for the specific detection of both synthase transcripts as well as of the housekeeping 16S rRNA, and the relative expression of target genes was calculated. We showed that phaC1 _Pc and phaC2 _Pc were not cotranscribed and, on the contrary, were independently regulated. In cultures grown with oleic acid as the sole carbon source, only the expression of phaC1 _Pc was induced (a tenfold increase after 72 h of culture), whereas that of phaC2 _Pc remained unchanged. In cultures grown with glucose or sodium octanoate, the expression of both phaC1 _Pc and phaC2 _Pc was upregulated but at different rates. Cellular PHA content was compared to the gene expression of the PHA synthases and significant correlations were found between PHA production and phaC1 _Pc/phaC2 _Pc expression.

Effect of culture conditions on mycelial growth, antibacterial activity, and metabolite profiles of the marine-derived fungus Arthrinium c.f. saccharicola by Li Miao; Theresa F. N. Kwong; Pei-Yuan Qian (pp. 1063-1073).

The effects of culture conditions and competitive cultivation with bacteria on mycelial growth, metabolite profile, and antibacterial activity of the marine-derived fungus Arthrinium c.f. saccharicola were investigated. The fungus grew faster at 30°C, at pH 6.5 and in freshwater medium, while exhibited higher antibacterial activity at 25°C, at pH 4.5, 5.5, and 7.5, and in 34 ppt seawater medium. The fungus grew faster in a high-nitrogen medium that contained 0.5% peptone and/or 0.5% yeast extract, while exhibiting higher bioactivity in a high-carbon medium that contained 2% glucose. The fungal growth was inhibited when it was co-cultured with six bacterial species, particularly the bacterium Pseudoalteromonas piscida. The addition of a cell free culture broth of this bacterium significantly increased the bioactivity of the fungus. Metabolite profiles of the fungus revealed by gas chromatography (GC)-mass spectrometry showed clear difference among different treatments, and the change of relative area of three peaks in GC profile followed a similar trend with the bioactivity variation of fungal extracts. Our results showed clear differences in the optimal conditions for achieving maximal mycelial growth and bioactivity of the fungus, which is important for the further study on the mass cultivation and bioactive compounds isolation from this fungus.

Development of a defined medium supporting rapid growth for Deinococcus radiodurans and analysis of metabolic capacities by Alexandra D. Holland; Heather M. Rothfuss; Mary E. Lidstrom (pp. 1074-1082).

A morpholinepropanesulfonic acid (MOPS)-buffered rich defined medium (RDM) was optimized to support a reproducible 2.6-h doubling time at 35 °C for Deinococcus radiodurans R1 and used to gain insight into vitamin and carbon metabolism. D. radiodurans was shown to require biotin and niacin for growth in this medium. A glutamine–serine simple defined medium (SDM) was developed that supported a 4-h doubling time, and this medium was used to probe sulfur and methionine metabolism. Vitamin B₁₂ was shown to alleviate methionine auxotrophy, and under these conditions, sulfate was used as the sole sulfur source. Phenotypic characterization of a methionine synthase deletion mutant demonstrated that the B₁₂ alleviation of methionine auxotrophy was due to the necessity of the B₁₂-dependent methionine synthase in methionine biosynthesis. Growth on ammonium as the sole nitrogen source in the presence of vitamin B₁₂ was demonstrated, but it was not possible to achieve reproducibly good growth in the absence of at least one amino acid as a nitrogen source. Growth on sulfate, cysteine, and methionine as sulfur sources demonstrated the function of a complete sulfur recycling pathway in this strain. These studies have demonstrated that rapid growth of D. radiodurans R1 can be achieved in a MOPS-based medium solely containing a carbon source, salts, four vitamins, and two amino acids.

Autecological properties of soil sphingomonads involved in the degradation of polycyclic aromatic hydrocarbons by Michael Cunliffe; Michael A. Kertesz (pp. 1083-1089).

Autecological properties that are thought to be important for polycyclic aromatic hydrocarbon (PAH)-degradation by bacteria in contaminated soils include the ability to utilize a broad range of carbon sources, efficient biofilm formation, cell-surface hydrophobicity, surfactant production, motility, and chemotaxis. Sphingomonas species are common PAH-degraders, and a selection of PAH-degrading sphingomonad strains isolated from contaminated soils was therefore characterized in terms of these properties. All the sphingomonads tested were relatively hydrophilic and were able to grow as biofilms on a phenanthrene-coated surface, though biofilm formation under other conditions was variable. Sphingobium yanoikuyae B1 was able to utilize the greatest range of carbon sources, though it was not chemotaxic towards any of the substrates tested. Other sphingomonad strains were considerably less flexible in their catabolic range. None of the strains produced detectable surfactant and swimming motility varied between the strains. Examination of the total Sphingomonas community in the soils tested showed that one of the isolates studied was present at significant levels, suggesting that it can thrive under PAH-contaminated conditions despite the lack of many of the tested characteristics. We conclude that these properties are not essential for survival and persistence of Sphingomonas in PAH-contaminated soils.

H₂S degradation is reflected by both the activity and composition of the microbial community in a compost biofilter by Bram Sercu; Nico Boon; Willy Verstraete; Herman Van Langenhove (pp. 1090-1098).

In this study, 16S rRNA- and rDNA-based denaturing gradient gel electrophoresis (DGGE) were used to study the temporal and spatial evolution of the microbial communities in a compost biofilter removing H₂S and in a control biofilter without H₂S loading. During the first 81 days of the experiment, the H₂S removal efficiencies always exceeded 93% at loading rates between 4.1 and 30 g m⁻³ h⁻¹. Afterwards, the H₂S removal efficiency decreased to values between 44 and 71%. RNA-based DGGE analysis showed that H₂S loading to the biofilter increased the stability of the active microbial community but decreased the activity-based diversity and evenness. The most intense band in both the RNA- and DNA-based DGGE patterns of the H₂S-degrading biofilter represented the sulfur oxidizing bacterium Thiobacillus thioparus. This suggested that T. thioparus constituted a major part of the bacterial community and was an important primary degrader in the H₂S-degrading biofilter. The decreasing H₂S removal efficiencies near the end of the experiment were not accompanied by a substantial change of the DGGE patterns. Therefore, the decreased H₂S removal was probably not caused by a failing microbiology but rather by a decrease of the mass transfer of substrates after agglutination of the compost particles.

Determination of soluble and granular inorganic polyphosphate in Corynebacterium glutamicum by Peter Klauth; Srinivas Reddy Pallerla; Dolores Vidaurre; Carla Ralfs; Volker F. Wendisch; Siegfried M. Schoberth (pp. 1099-1106).

Corynebacterium glutamicum forms inorganic polyphosphate (poly P) that may occur as soluble (cytosolic) poly P and/or as volutin granules. A suitable method for monitoring soluble and granular poly P in C. glutamicum was developed and applied to C. glutamicum cells cultivated under different growth conditions. Under phosphate-limiting conditions, C. glutamicum did not accumulate poly P, but it rebuilt its poly P storages when phosphate became available. The poly P content of C. glutamicum growing on glucose minimal medium with sufficient phosphate varied considerably during growth. While the poly P content was minimal in the midexponential growth phase, two maxima were observed in the early exponential growth phase and at entry into the stationary growth phase. Cells in the early exponential growth phase primarily contained granular poly P, while cells entering the stationary growth phase contained soluble, cytosolic poly P. These results and those obtained for C. glutamicum cells cultivated under hypo- or hyperosmotic conditions or during glutamate production revealed that the poly P content of C. glutamicum and the partitioning between cytosolic and granular forms of poly P are dynamics and depend on the growth conditions.

Keywords: Poly P; Volutin granules; DAPI; ³¹P NMR; Phosphate starvation; Osmoshock; Ethambutol; Tween 40; Glutamate production; Digital image analysis; Corynebacterium glutamicum

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Applied Microbiology and Biotechnology (v.72, #5)

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Applied Microbiology and Biotechnology (v.72, #5)