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Applied Microbiology and Biotechnology (v.75, #6)

Protein engineering: opportunities and challenges by Matti Leisola; Ossi Turunen (pp. 1225-1232).

The extraordinary properties of natural proteins demonstrate that life-like protein engineering is both achievable and valuable. Rapid progress and impressive results have been made towards this goal using rational design and random techniques or a combination of both. However, we still do not have a general theory on how to specify a structure that is suited to a target function nor can we specify a sequence that folds to a target structure. There is also overreliance on the Darwinian blind search to obtain practical results. In the long run, random methods cannot replace insight in constructing life-like proteins. For the near future, however, in enzyme development, we need to rely on a combination of both.

Keywords: Protein engineering; Directed evolution; Enzymes

Application of DNA methyltransferases in targeted DNA methylation by Albert Jeltsch; Renata Z. Jurkowska; Tomasz P. Jurkowski; Kirsten Liebert; Philipp Rathert; Martina Schlickenrieder (pp. 1233-1240).

DNA methylation is an essential epigenetic modification. In bacteria, it is involved in gene regulation, DNA repair, and control of cell cycle. In eukaryotes, it acts in concert with other epigenetic modifications to regulate gene expression and chromatin structure. In addition to these biological roles, DNA methyltransferases have several interesting applications in biotechnology, which are the main focus of this review, namely, (1) in vivo footprinting: as several bacterial DNA methyltransferases cannot methylate DNA bound to histone proteins, the pattern of DNA methylation after expression of DNA methyltransferases in the cell allows determining nucleosome positioning; (2) mapping the binding specificity of DNA binding proteins: after fusion of a DNA methyltransferase to a DNA-binding protein and expression of the fusion protein in a cell, the DNA methylation pattern reflects the DNA-binding specificity of the DNA-binding protein; and (3) targeted gene silencing: after fusion of a DNA methyltransferase to a suitable DNA-binding domain, DNA methylation can be directed to promoter regions of target genes. Thereby, gene expression can be switched off specifically, efficiently, and stably, which has a number of potential medical applications.

Keywords: DNA methyltransferase; Epigenetics; Enzyme design; DamID

Biosynthesis, properties and potential of natural–synthetic hybrids of polyhydroxyalkanoates and polyethylene glycols by L. John R. Foster (pp. 1241-1247).

Chemical conjugation with poly(ethylene glycols) (PEGs) are established procedures to facilitate solubilisation of hydrophobic compounds. Such techniques for PEGylation have been applied to polyhydroxybutyrate. ‘BioPEGylation’ of such polyhydroxyalkanoates (PHAs) to form natural–synthetic hybrids has been demonstrated through the addition of PEGs to microbial cultivation systems. The strategic addition of certain PEGs not only supports hybrid synthesis but may also provide a technique for control of PHA composition and molecular mass, and by extension, their physico-mechanical properties. PHA composition and molecular mass control by PEGs is dependent upon the polyethers’ molecular mass, loading in the cultivation system, time of introduction and microbial species. Hybrid characterisation studies are in their infancy, but results to date suggest that PHA–PEG hybrids have subtle, but significant, differences in their physiochemical and material properties as a consequence of the PEGylation.

Keywords: Polyhydroxyalkanoates; Poly(ethylene glycol); PEGylation; Biomaterials; Molecular weight; Bioprocessing

Continuous production of chiral 1,3-butanediol using immobilized biocatalysts in a packed bed reactor: promising biocatalysis method with an asymmetric hydrogen-transfer bioreduction by Nobuya Itoh; Masatoshi Nakamura; Kousuke Inoue; Yoshihide Makino (pp. 1249-1256).

An asymmetric hydrogen-transfer biocatalyst consisting of mutated Rhodococcus phenylacetaldehyde reductase (PAR) or Leifsonia alcohol dehydrogenase (LSADH) was applied for some water-soluble ketone substrates. Among them, 4-hydroxy-2-butanone was reduced to (S)/(R)-1,3-butanediol, a useful intermediate for pharmaceuticals, with a high yield and stereoselectivity. Intact Escherichia coli cells overexpressing mutated PAR (Sar268) or LSADH were directly immobilized with polyethyleneimine or 1,6-diaminehexane and glutaraldehyde and evaluated in a batch reaction. This system produced (S)-1,3-butanediol [87% enantiomeric excess (e.e.)] with a space time yield (STY) of 12.5 mg h⁻¹ ml⁻¹ catalyst or (R)-1,3-butanediol (99% e.e.) with an STY of 60.3 mg h⁻¹ ml⁻¹ catalyst, respectively. The immobilized cells in a packed bed reactor continuously produced (R)-1,3-butanediol with a yield of 99% (about 49.5 g/l) from 5% (w/v) 4-hydroxy-2-butanoate over 500 h.

Keywords: Chiral 1, 3-butanediol; Biocatalyst; Asymmetric reduction; 2-propanol; Hydrogen-transfer

Hypoglycemic effects of exopolysaccharides produced by mycelial cultures of two different mushrooms Tremella fuciformis and Phellinus baumii in ob/ob mice by Eun Jae Cho; Hye Jin Hwang; Sang Woo Kim; Jung Young Oh; Yu Mi Baek; Jang Won Choi; Song Hwan Bae; Jong Won Yun (pp. 1257-1265).

The anti-diabetic activities of the exopolysaccharides (EPS) produced by submerged mycelial culture of two different mushrooms, Tremella fuciformis and Phellinus baumii, in ob/ob mice were investigated. All the animals were randomly divided into three groups with seven animals in each group: The control group received 0.9% NaCl solution; the diabetic groups were treated with EPS from T. fuciformis (Tf EPS) and P. baumii (Pb EPS) at the level of 200 mg/kg body weight using an oral zoned daily for 52 days. The plasma glucose levels in the EPS-fed mice were substantially reduced by about 52% (Tf EPS) and 32% (Pb EPS), respectively, as compared to control mice. The results of oral glucose tolerance test (OGTT) revealed that both EPS-fed groups significantly increased the glucose disposal after 52 days of EPS treatments. Furthermore, higher food efficiency ratios and reduced blood triglyceride levels were observed in the EPS-treated groups. Because peroxisome proliferator-activated receptor gamma (PPAR-γ) is indeed a key regulator of insulin action, we investigated the expression pattern of adipose tissue PPAR-γ messenger RNA (mRNA) and plasma levels of PPAR-γ. It was revealed that PPAR-γ was significantly activated in response to EPS treatments. The results suggested that both EPS exhibited considerable hypoglycemic effect and improved insulin sensitivity possibly through regulating PPAR-γ-mediated lipid metabolism. Our results indicated that two mushroom-derived EPS might be developed as potential oral hypoglycemic agents or functional foods for the management of non-insulin-dependent diabetes mellitus.

Keywords: Exopolysaccharides; Diabetes mellitus; Hypoglycemic effect; ob/ob mice; Phellinus baumii ; Tremella fuciformis

Enhanced iturin A production by Bacillus subtilis and its effect on suppression of the plant pathogen Rhizoctonia solani by S. Mizumoto; M. Hirai; M. Shoda (pp. 1267-1274).

Enhanced production of the antibiotic iturin A by Bacillus subtilis RB14-CS reached 4.4 g L⁻¹ in SM medium containing soybean meal and maltose, which was 16-fold and 2.2-fold higher than that in original and modified number 3S media, respectively. When various volumes of RB14-CS cultures grown in SM medium were applied to pot tests of tomato damping-off caused by Rhizoctonia solani, damping-off was dose-dependently suppressed by the cultures. Suppression by SM-grown cultures was significantly more effective than that by cultures grown in original or modified number 3S media. The iturin A concentrations in soil decreased to undetectable levels after 17 days of cultivation in pot tests, indicating that iturin A has a low persistence in soil.

Characterization of a chitinolytic enzyme from Serratia sp. KCK isolated from kimchi juice by Hyun-Soo Kim; Kenneth N. Timmis; Peter N. Golyshin (pp. 1275-1283).

The novel chitinolytic bacterium Serratia sp. KCK, which was isolated from kimchi juice, produced chitinase A. The gene coding for the chitinolytic enzyme was cloned on the basis of sequencing of internal peptides, homology search, and design of degenerated primers. The cloned open reading frame of chiA encodes for deduced polypeptide of 563 amino acid residues with a calculated molecular mass of 61 kDa and appears to correspond to a molecular mass of about 57 kDa, which excluded the signal sequence. The deduced amino acid sequence showed high similarity to those of bacterial chitinases classified as family 18 of glycosyl hydrolases. The chitinase A is an exochitinase and exhibits a greater pH range (5.0–10.0), thermostability with a temperature optimum of 40°C, and substrate range other than Serratia chitinases thus far described. These results suggested that Serratia sp. KCK chitinase A can be used for biotechnological applications with good potential.

Keywords: Chitinolytic bacteria and enzyme; Chitinase A; Kimchi juice

Metallopeptidase, neurolysin, as a novel molecular tool for analysis of properties of cancer-producing matrix metalloproteinases-2 and -9 by Tetsuya Kadonosono; Michiko Kato; Mitsuyoshi Ueda (pp. 1285-1291).

To compare the substrate preferences of rat brain neurolysin and cancer-producing matrix metalloproteinases (MMPs), which have the same architecture in their catalytic domains, the cleavage activity of neurolysin toward MMP-specific fluorescence-quenching peptides was quantitatively measured. The results show that neurolysin effectively cleaved MOCAc [(7-methoxy coumarin-4-yl) acetyl]-RPKPYANvaWMK(Dnp[2,4-dinitrophenyl])-NH₂, a specific substrate of MMP-2 and MMP-9, but hardly cleaved MOCAc-RPKPVENvaWRK(Dnp)-NH₂, a specific substrate of MMP-3, suggesting that neurolysin has a similar substrate preference to MMP-2 and MMP-9. A structural comparison between neurolysin and MMP-9 showed the similar key amino acid residues for substrate recognition. The possible application of neurolysin displayed on the yeast cell surface, as a safe protein alternative to MMP-2 and MMP-9 which induce cancer cell growth, invasion, and metastasis, to analysis of properties of the MMPs, including the screening of inhibitors and analysis of inhibition mechanism etc., are also discussed.

Keywords: Molecular display; Matrix metalloproteinase; Neurolysin; Substrate preference; Specific inhibitors

Structural characterization of heterodimeric laccases from Pleurotus ostreatus by Paola Giardina; Flavia Autore; Vincenza Faraco; Giovanna Festa; Gianna Palmieri; Alessandra Piscitelli; Giovanni Sannia (pp. 1293-1300).

The subfamily of POXA3 laccase isoenzymes produced by the fungus Pleurotus ostreatus has been characterized as an example of the complexity and heterogeneity of fungal isoenzyme patterns. Two isoenzymes, POXA3a and POXA3b, were previously purified, exhibiting an unusual heterodimeric structure composed of a large (67 kDa) and a small (18 or 16 kDa) subunit. A unique gene encodes the large subunit of both POXA3a and POXA3b, but alternative splicing produces two variants—differing for an insertion of four amino acids—for each isoenzyme. Two genes encoding POXA3a and POXA3b small subunits have been identified, and the corresponding amino acid sequences show only two amino acid substitutions. The 18- and 16-kDa subunits of both POXA3a and POXA3b differ for N-glycosylation at Asn150 of the 16-kDa subunit. The POXA3 large subunit 3D model allows us to highlight peculiarities of this molecule with respect to the laccases whose 3D structures are known.

Keywords: Phenol oxidase; White rot fungi; Quaternary structure

Stable and continuous long-term enzymatic reaction using an enzyme–nanofiber composite by Jin Hyung Lee; Ee Taek Hwang; Byoung Chan Kim; Sun-Mi Lee; Byoung-In Sang; Yong Su Choi; Jungbae Kim; Man Bock Gu (pp. 1301-1307).

This study shows the preparation and application of enzyme–nanofiber composites for long-term stable operation. The enzyme–nanofiber composite was prepared by coating an enzyme aggregate, the esterase from Rhizopus oryzae, on the surface of the nanofibers. After immobilization on the nanofiber, the apparent K m for the immobilized esterase was 1.48-fold higher than that of the free esterase, with values of 0.98 and 1.35 mM for the free and immobilized enzymes, respectively. It was found that enzyme–nanofiber was very stable, even when the fibers were shaken in glass vials, preserving 80% of the initial activity for 100 days. In addition, the enzyme–nanofiber composite was used repeatedly in 30 cycles of substrate hydrolysis and still remained active. Consequently, the esterase–nanofiber composite was employed within a continuous reactor system to evaluate its use in a long-term and stable continuous substrate hydrolysis reaction. It was found that the production of p-nitrophenol was stable for at least 400 h. This study demonstrates that the enzyme–nanofiber composite can be used in both repeated-batch mode and a continuous mode for a long-term stable operation.

Keywords: Continuous reactor; Nanofiber; Esterase; Enzyme stabilization; Enzyme immobilization; Enzyme reactor

Recombinant expression and characterization of XynD from Bacillus subtilis subsp. subtilis ATCC 6051: a GH 43 arabinoxylan arabinofuranohydrolase by Tine M. Bourgois; Valerie Van Craeyveld; Steven Van Campenhout; Christophe M. Courtin; Jan A. Delcour; Johan Robben; Guido Volckaert (pp. 1309-1317).

The complete genome sequence of Bacillus subtilis reveals that sequences encoding several hemicellulases are co-localised with a gene (xynD) encoding a putative family 43 glycoside hydrolase that has not yet been characterised. In this work, xynD has been isolated from genomic DNA of B. subtilis subsp. subtilis ATCC 6051 and cloned for cytoplasmatic expression in Escherichia coli. Recombinant XynD (rXynD) was purified using ion-exchange chromatography and gel permeation chromatography. The enzyme had a molecular mass of approximately 52 kDa, a pI above 9.0 and releases α-l-arabinose from arabinoxylo-oligosaccharides as well as arabinoxylan polymers with varying degree of substitution. Using para-nitrophenyl-α-l-arabinofuranoside as substrate, maximum activity was observed at pH 5.6 and 45°C. The enzyme retained its activity over a large pH range, while activity was lost after pre-incubation above 50°C. Gas–liquid chromatography and proton nuclear magnetic resonance spectrometry analysis indicated that rXynD specifically releases arabinofuranosyl groups from mono-substituted C-(O)-2 and C-(O)-3 xylopyranosyl residues on the xylan backbone. As rXynD did not display endoxylanase, xylosidase or arabinanase activity and was inactive on arabinan, we conclude that this enzyme is best described as an arabinoxylan arabinofuranohydrolase.

Keywords: Bacillus subtilis ; Arabinoxylan arabinofuranohydrolase; Glycoside hydrolase family 43; Arabinoxylan-derived oligosaccharides; α-l-arabinose

Development of yeast cells displaying Candida antarctica lipase B and their application to ester synthesis reaction by Takanori Tanino; Takumi Ohno; Tohru Aoki; Hideki Fukuda; Akihiko Kondo (pp. 1319-1325).

We isolated the lipase B from Candida antarctica CBS 6678 (CALB CBS6678) and successfully constructed CALB-displaying yeast whole-cell biocatalysts using the Flo1p short (FS) anchor system. For the display of CALB on a yeast cell surface, the newly isolated CALB CBS6678 exhibited higher hydrolytic and ester synthesis activities than the well-known CALB, which is registered in GenBank (Z30645). A protease accessibility assay using papain as a protease showed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface. A comparison of the lipase hydrolytic activities of yeast whole cells displaying only mature CALB (CALB) and those displaying mature CALB with a Pro region (ProCALB) revealed that mature CALB is preferable for yeast cell surface display using the Flo1p anchor system. Lyophilized yeast whole cells displaying CALB were applied to an ester synthesis reaction at 60°C using adipic acid and n-butanol as substrates. The amount of dibutyl adipate (DBA) produced increased with the reaction time until 144 h. This indicated that CALB displayed on the yeast cell surface retained activity under the reaction conditions.

Keywords: Cell surface display; Candida antarctica lipase B; Ester synthesis

Molecular and biochemical characterization of Ba-EGA, a cellulase secreted by Bacillus sp. AC-1 from Ampullaria crosseans by Shuang Zhang; Qiu-yu Yin; Yan-hong Li; Ming Ding; Gen-jun Xu; Fu-kun Zhao (pp. 1327-1334).

A novel gene (Ba-ega) of Bacillus sp. AC-1, encoding an endoglucanase (Ba-EGA), was cloned and expressed in Escherichia coli. Ba-ega, containing a 1,980-bp open reading frame (ORF), encoded a protein of 659 amino acids and had a molecular mass of 74.87 kDa. Ba-EGA was a modular enzyme composed of a family-9 glycosyl hydrolase catalytic module (CM9) and a family-3 carbohydrate-binding module (CBM3). To investigate the functions of the CBM3 and CM9, a number of truncated derivatives of Ba-EGA were constructed, and all were active. The catalytic module (rCM9) alone was less stable at high temperature than the recombinant Ba-EGA (rBa-EGA). The temperature stability for the complex of rCM9 and rCBM3 was still lower than rBa-EGA, but higher than rCM9 alone. These observations indicated the existence of a non-covalent interaction between CM9 and CBM3 that might strengthen the stability of CM9. However, this interaction is not strong enough to mimic the protective effect of the CBM in the wild-type enzyme.

Keywords: Gene cloning; Expression; Carbohydrate binding module; Non-covalent interactions

Characterization of two β-carotene ketolases, CrtO and CrtW, by complementation analysis in Escherichia coli by Seon-Kang Choi; Hisashi Harada; Satoru Matsuda; Norihiko Misawa (pp. 1335-1341).

The pathways from β-carotene to astaxanthin are crucial key steps for producing astaxanthin, one of industrially useful carotenoids, in heterologous hosts. Two β-carotene ketolases (β-carotene 4,4′-oxygenase), CrtO and CrtW, with different structure are known up to the present. In this paper, we compared the catalytic functions of a CrtO ketolase that was obtained from a marine bacterium Rhodococcus erythropolis strain PR4, CrtO derived from cyanobacterium Synechosistis sp. PCC6803, and CrtW derived from a marine bacterium Brevundimonas sp. SD212, by complementation analysis in Escherichia coli expressing the known crt genes. Results strongly suggested that a CrtO-type ketolase was unable to synthesize astaxanthin from zeaxanthin, i.e., only a CrtW-type ketolase could accept 3-hydroxy-β-ionone ring as the substrate. Their catalytic efficiency for synthesizing canthaxanthin from β-carotene was also examined. The results obtained up to the present clearly suggest that the bacterial crtW and crtZ genes are a combination of the most promising gene candidates for developing recombinant hosts that produce astaxanthin as the predominant carotenoid.

Keywords: Astaxanthin; Carotenoid; CrtW; CrtO

N-Acetyltransferase Mpr1 confers ethanol tolerance on Saccharomyces cerevisiae by reducing reactive oxygen species by Xiaoyi Du; Hiroshi Takagi (pp. 1343-1351).

N-Acetyltransferase Mpr1 of Saccharomyces cerevisiae can reduce intracellular oxidation levels and protect yeast cells under oxidative stress, including H₂O₂, heat-shock, or freeze-thaw treatment. Unlike many antioxidant enzyme genes induced in response to oxidative stress, the MPR1 gene seems to be constitutively expressed in yeast cells. Based on a recent report that ethanol toxicity is correlated with the production of reactive oxygen species (ROS), we examined here the role of Mpr1 under ethanol stress conditions. The null mutant of the MPR1 and MPR2 genes showed hypersensitivity to ethanol stress, and the expression of the MPR1 gene conferred stress tolerance. We also found that yeast cells exhibited increased ROS levels during exposure to ethanol stress, and that Mpr1 protects yeast cells from ethanol stress by reducing intracellular ROS levels. When the MPR1 gene was overexpressed in antioxidant enzyme-deficient mutants, increased resistance to H₂O₂ or heat shock was observed in cells lacking the CTA1, CTT1, or GPX1 gene encoding catalase A, catalase T, or glutathione peroxidase, respectively. These results suggest that Mpr1 might compensate the function of enzymes that detoxify H₂O₂. Hence, Mpr1 has promising potential for the breeding of novel ethanol-tolerant yeast strains.

Keywords: N-Acetyltransferase Mpr1; Ethanol tolerance; Oxidative stress; Reactive oxygen species; Saccharomyces cerevisiae

Substrate specificity of rat brain neurolysin disclosed by molecular display system and putative substrates in rat tissues by Tetsuya Kadonosono; Michiko Kato; Mitsuyoshi Ueda (pp. 1353-1360).

To search for the substrates, other than neurotensin, of rat brain neurolysin, a novel method of determining peptidase activity was developed using a yeast molecular display system. This is a useful and convenient method of handling homogenously pure proteins to evaluate the properties of neurolysin. The neurolysin gene was ligated to the C-terminal half of the α-agglutinin gene with a FLAG tag sequence and a yeast cell-surface molecular displaying plasmid was constructed. Display of neurolysin with correct folding and appropriate activity was verified by immunofluorescence staining and activity measurement of a bradykinin-related peptide. The cleavage sites of peptides were determined by high-performance liquid chromatography (HPLC) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results showed the amino acid preferences of hydrophobic, aromatic, and basic residues, which were the same as those of soluble neurolysin. Moreover, this method clearly showed the presence of two recognition motifs in neurolysin. By using these motifs, novel substrate candidates of neurolysin in rat tissues were screened, and several bioactive peptides that regulate feeding were found. We also discussed the ubiquitous distribution of neurolysin in rat tissues and the functions of substrate candidate peptides.

Keywords: Molecular display; Neurolysin; Cell surface display; Rat brain; Peptidase

Over-expressing GLT1 in a gpd2Δ mutant of Saccharomyces cerevisiae to improve ethanol production by Qing-Xue Kong; Ai-Li Zhang; Li-Min Cao; Xun Chen (pp. 1361-1366).

We constructed two recombinant strains of Saccharomyces cerevisiae in which the GPD2 gene was deleted using a one-step gene replacement method to minimize formation of glycerol and improve ethanol production. In addition, we also over-expressed the GLT1 gene by a two-step gene replacement method to overcome the redox-imbalancing problem in the genetically modified strains. The result of anaerobic batch fermentations showed that the rate of growth and glucose consumption of the KAM-5 (MATα ura3 gpd2Δ::RPT) strain were slower than the original strain, and the KAM-13 (MATα ura3 gpd2Δ::RPT P PGK -GLT1) strain, however, was indistinguishable compared to the original strain using the same criteria, as analyzed. On the other hand, when compared to the original strain, there were 32 and 38% reduction in glycerol formation for KAM-5 and KAM-13, respectively. Ethanol production increased by 8.6% for KAM-5 and 13.4% for KAM-13. Dramatic reduction in acetate and pyruvic acid was also observed in both mutants compared to the original strains. Although gene GPD2 is responsible for the glycerol synthesis, the mutant KAM-13, in which glycerol formation was substantially reduced, was able to cope and maintain osmoregulation and redox balance and have increased ethanol production under anaerobic fermentations. The result verified the proposed concept of increasing ethanol production in S. cerevisiae by genetic engineering of glycerol synthesis and over-expressing the GLT1 gene along with reconstituted nicotinamide adenine dinucleotide metabolism.

Keywords: Ethanol yield; Glycerol formation; Glutamate-synthase gene; Glycerol 3-phosphate dehydrogenase; S. cerevisiae

A strategy for cloning glycosyltransferase genes involved in natural product biosynthesis by Andriy Luzhetskyy; Holger Weiss; Annette Charge; Elisabeth Welle; Anton Linnenbrink; Andreas Vente; Andreas Bechthold (pp. 1367-1375).

The soil-borne and marine gram-positive Actinomycetes are a particularly rich source of carbohydrate-containing metabolites. With the advent of molecular tools and recombinant methods applicable to Actinomycetes, it has become feasible to investigate the biosynthesis of glycosylated compounds at genetic and biochemical levels, which has finally set the basis for engineering novel natural product derivatives. Glycosyltransferases (GT) are key enzymes for the biosynthesis of many valuable natural products that contain sugar moieties and they are most important for drug engineering. So far, the direct cloning of unknown glycosyltransferase genes by polymerase chain reaction (PCR) has not been described because glycosyltransferases do not share strongly conserved amino acid regions. In this study, we report a method for cloning of novel so far unidentified glycosyltransferase genes from different Actinomycetes strain. This was achieved by designing primers after a strategy named consensus-degenerate hybrid oligonucleotide primer (CODEHOP). Using this approach, 22 novel glycosyltransferase encoding genes putatively involved in the decoration of polyketides were cloned from the genomes of 10 Actinomycetes. In addition, a phylogenetic analysis of glycosyltransferases from Actinomycetes is shown in this paper.

Keywords: Hybrid primers; CODEHOP; Natural products; Glycosyltransferase evolution

Functional analysis of genes involved in the biosynthesis of isoprene in Bacillus subtilis by Mattijs K. Julsing; Michael Rijpkema; Herman J. Woerdenbag; Wim J. Quax; Oliver Kayser (pp. 1377-1384).

In comparison to other bacteria Bacillus subtilis emits the volatile compound isoprene in high concentrations. Isoprene is the smallest representative of the natural product group of terpenoids. A search in the genome of B. subtilis resulted in a set of genes with yet unknown function, but putatively involved in the methylerythritol phosphate (MEP) pathway to isoprene. Further identification of these genes would give the possibility to engineer B. subtilis as a host cell for the production of terpenoids like the valuable plant-produced drugs artemisinin and paclitaxel. Conditional knock-out strains of putative genes were analyzed for the amount of isoprene emitted. Differences in isoprene emission were used to identify the function of the enzymes and of the corresponding selected genes in the MEP pathway. We give proof on a biochemical level that several of these selected genes from this species are involved in isoprene biosynthesis. This opens the possibilities to investigate the physiological function of isoprene emission and to increase the endogenous flux to the terpenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate, for the heterologous production of more complex terpenoids in B. subtilis.

Keywords: Bacillus subtilis ; Isoprene; Methylerythritol phosphate pathway; MEP pathway; Terpenoid; Gas chromatography

Enhanced expression, detection and purification of recombinant proteins using RNA stem loop and tandem fusion tags by Mohamed Seleem; Mohammed Ali; M. W. Abd Al-Azeem; Stephen M. Boyle; Nammalwar Sriranganathan (pp. 1385-1392).

The creation of a double His-tag fusion that forms a RNA stem loop in the mRNA encoding the N-terminus of the target protein is a novel approach for the enhancement of expression, purification, and detection of a recombinant protein. Compared to a single His-tag fusion, a tandem His-tag fusion RNA stem loop, located downstream of the constitutive groE and Ch promoters, enhanced heterologous gene expression in Brucella, Salmonella, and Escherichia. We demonstrated one-step detection and purification of recombinant green fluorescence protein (GFP) directly from Brucella spp. without using Escherichia coli as an expression host. The amount of purified GFP using the tandem His-tag RNA stem loop increased more than threefold; moreover, the sensitivity of detection increased more than fourfold in comparison to the single His-tag fusion form. This method has the potential to significantly improve heterologous gene expression and high-throughput protein synthesis and purification.

Keywords: RNA stem loop; His tag; Protein; Purification; Brucella ; Vector; Gene expression

Release of thioredoxin from Saccharomyces cerevisiae with environmental stimuli: solubilization of thioredoxin with ethanol by Yoko Takeuchi; Wataru Nomura; Takumi Ohdate; Shogo Tamasu; Hiroshi Masutani; Kazuo Murata; Shingo Izawa; Junji Yodoi; Yoshiharu Inoue (pp. 1393-1399).

Thioredoxin is crucial for the maintenance of the redox status of cells of all types. Mammalian thioredoxin is secreted from various types of cells, although the mechanism underlying has not yet been clarified. Previously, we demonstrated that thioredoxin was released from Saccharomyces cerevisiae after treatment with ethanol. In this paper, we show that as well as ethanol, low-pH shock and hypoosmotic shock release thioredoxin. Low-molecular-weight proteins in yeast cells were preferentially released by treatment with ethanol and low-pH shock. A cell wall integrity pathway seems partially involved in the hypoosmotic shock-induced release of thioredoxin. Considerable amounts of thioredoxin were present in the insoluble fractions of the cells, a portion of which was associated with lipid microdomains that are resistant to nonionic detergent at 4°C. The intracellular localization of thioredoxin may influence the efficiency of its release from yeast cells with ethanol.

Screening of high-yielding biocontrol bacterium Bs-916 mutant by ion implantation by Dequan Li; Fengya Nie; Lihui Wei; Benqiang Wei; Zhiyi Chen (pp. 1401-1408).

Bacillus subtilis 916 was an effective biocontrol agent in control rice sheath blight caused by Rhizoctonia solani. To further improve its antagonistic ability, low-energy ion implantation was applied in Bs-916. We studied the effects of different doses of N⁺ implantation. The optimum dose of ion implantation for the Bs-916 was from 15 × 2.6 × 10¹⁴ N⁺/cm² to 25 × 2.6 × 10¹⁴ N⁺/cm². The mutant strain designated as Bs-H74 was obtained, which showed higher inhibition activity in the screening plate. Its inhibition zone against the indicator organism increased by 30.7% compared to the parental strain. The control effect of rice sheath blight was improved by 14.6% over that of Bs-916. Thin-layer chromatography and high-performance liquid chromatography analysis indicated that lipopeptides produced by Bs-916 and the mutant strains belonged to the surfactin family. Bs-H74 produced approximately 3.0-fold surfactin compared to that of Bs-916. To determine the role of surfactin in biocontrol by Bs-916, we tested another mutant strain, Bs-M49, which produced lower levels of surfactin significantly, and found that Bs-M49 had no obvious effects against R. solani. These results suggested that the surfactin produced by Bs-916 plays an important role in the suppression of sheath blight. These observations also showed that the Bs-H74 mutant strain is a better biocontrol agent than the parental strain.

Keywords: Screening; High-yielding mutant; Ion Implantation

Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica by André Förster; Andreas Aurich; Stephan Mauersberger; Gerold Barth (pp. 1409-1417).

The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0–6.8 and resulted in production of 127–140 g l⁻¹ CA with a yield Y _CA of 0.75–0.82 g g⁻¹, whereas at pH 5.0, 87 g l ⁻¹ with a yield Y _CA of 0.51 gg⁻¹ were produced. The CA-productivity Q _CA increased from 0.40 g l ⁻¹ h⁻¹ at pH 5.0 up to 0.73 g l ⁻¹ h⁻¹ at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene–dose-dependent high isocitrate lyase expression resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production.

Keywords: Yarrowia lipolytica ; Sucrose; Invertase; Citric acid; Product selectivity

Differential role of microenvironment in microencapsulation for improved cell tolerance to stress by Zhi-jie Sun; Guo-jun Lv; Shuang-yue Li; Wei-ting Yu; Wei Wang; Yu-bing Xie; Xiaojun Ma (pp. 1419-1427).

The effect of the microenvironment in alginate–chitosan–alginate (ACA) microcapsules with liquid core (LCM) and solid core (SCM) on the physiology and stress tolerance of Sacchromyces cerevisiae was studied. The suspended cells were used as control. Cells cultured in liquid core microcapsules showed a nearly twofold increase in the intracellular glycerol content, trehalose content, and the superoxide dismutase (SOD) activity, which are stress tolerance substances, while SCM did not cause the significant physiological variation. In accordance with the physiological modification after being challenged with osmotic stress (NaCl), oxidative stress (H₂O₂), ethanol stress, and heat shock stress, the cell survival in LCM was increased. However, SCM can only protect the cells from damaging under ethanol stress. Cells released from LCM were more resistant to hyperosmotic stress, oxidative stress, and heat shock stress than cells liberated from SCM. Based on reasonable analysis, a method was established to estimate the effect of microenvironment of LCM and SCM on the protection of cells against stress factors. It was found that the resistance of LCM to hyperosmotic stress, oxidative stress, and heat shock stress mainly depend on the domestication effect of LCM’s microenvironment. The physical barrier of LCM constituted by alginate–chitosan membrane and liquid alginate matrix separated the cells from the damage of oxidative stress and ethanol stress. The significant tolerance against ethanol stress of SCM attributed to the physical barrier consists of solid alginate–calcium matrix and alginate–chitosan membrane.

Keywords: Sacchromyces cerevisiae ; ACA microcapsules; Microenvironment; Stress tolerance

Bioreduction of Pt (IV) from aqueous solution using sulphate-reducing bacteria by K. J. Rashamuse; C. G. Whiteley (pp. 1429-1435).

The aims of this study were to investigate the role of sulphate-reducing bacteria in facilitating Pt removal from aqueous solutions and to investigate the role of a hydrogenase enzyme in Pt reduction in vitro. To avoid precipitation of Pt as Pt sulphide, a resting (non-growing) mixed culture was used. A pH-dependent rate of Pt removal from aqueous solution was observed, indicating that metal speciation was the main factor for its removal from solution. The maximum initial concentration of Pt(IV) that the cells can effectively remove from solution was 50 mg/l, while the maximum capacity was only 4 mg of Pt per gram of resting biomass. Transmission electron microscopy and energy dispersive X-ray analyses indicated that Pt was being precipitated in the periplasm, a major area of hydrogenase activity in the cells. In vitro investigation of Pt reduction with hydrogen as the electron donor showed that 49% was removed within 1 h when a relatively pure hydrogenase extract was used, 31% was removed with a cell-free soluble extract and 70% removed by live cells.

Keywords: Sulphate-reducing bacteria; Hydrogenase; Bioremediation of platinum

Development of an efficient method for screening microorganisms by using symbiotic association between Nasutitermes takasagoensis and intestinal microorganisms by Arata Hayashi; Hideki Aoyagi; Kazuhiko Kinjyo; Tsuyoshi Yoshimura; Hideo Tanaka (pp. 1437-1446).

Screening method of microorganisms that utilized the symbiotic association between insect (Nasutitermes takasagoensis: Nt) and intestinal microorganisms was developed. The existence of desired microorganisms that grew by degrading difficult-to-degrade materials in the gut was detected using survivability of Nt as an indicator. The desired microorganisms were isolated from the survived Nt. It was thought that guts of Nt behave as continuous culture systems whereby microorganisms that cannot degrade diet components are washed out, whereas those that can degrade it are retained and concentrated in the gut. About 60% of Nt fed with phenol artificial diet (PAD) died within 7 days, while 4% of termites survived for 9 days. The structure of intestinal microorganisms of the survived Nt fed with PAD differed from the bacterial communities obtained from enrichment culture (which contained phenol) of wood-feeding Nt. Relatively high colonies (650-times) were detected in the gut of Nt fed on phenol artificial diet compared with those obtained when Nt was fed on wood. Seven denaturing gradient gel electrophoresis (DGGE) bands were detected from gut of wood-feeding Nt, whereas 11 DGGE-bands were detected from that of phenol-feeding Nt. Out of 11 DGGE-bands, 5 of them were sequenced, and bacterial species including phenol-degrading bacteria were identified.

Keywords: Screening method; Insect; Nasutitermes takasagoensis ; Artificial diet; Intestinal microorganisms

Comparison of volatile sulphur compound production by cheese-ripening yeasts from methionine and methionine–cysteine mixtures by M. López del Castillo-Lozano; A. Delile; H. E. Spinnler; P. Bonnarme; S. Landaud (pp. 1447-1454).

Production of volatile sulphur compounds (VSC) was assessed in culture media supplemented with l-methionine or l-methionine/l-cysteine mixtures, using five cheese-ripening yeasts: Debaryomyces hansenii DH47(8), Kluyveromyces lactis KL640, Geotrichum candidum GC77, Yarrowia lipolytica YL200 and Saccharomyces cerevisiae SC45(3). All five yeasts produced VSC with l-methionine or l-methionine/l-cysteine, but different VSC profiles were found. GC77 and YL200 produced dimethyldisulphide and trace levels of dimethyltrisulphide while DH47(8), KL640 and SC45(3) produced mainly methionol and low levels of methional. S-methylthioacetate was produced by all the yeasts but at different concentrations. DH47(8), KL640 and SC45(3) also produced other minor VSC including 3-methylthiopropyl acetate, ethyl-3-methylthiopropanoate, a thiophenone, and an oxathiane. However, VSC production diminished in a strain-dependent behaviour when l-cysteine was supplemented, even at a low concentration (0.2 g l⁻¹). This effect was due mainly to a significant decrease in l-methionine consumption in all the yeasts except YL200. Hydrogen sulphide produced by l-cysteine catabolism did not seem to contribute to VSC generation at the acid pH of yeast cultures. The significance of such results in the cheese-ripening context is discussed.

Vertical migration of aggregated aerobic and anaerobic ammonium oxidizers enhances oxygen uptake in a stagnant water layer by Siegfried E. Vlaeminck; Katleen Dierick; Nico Boon; Willy Verstraete (pp. 1455-1461).

Ammonium can be removed as dinitrogen gas by cooperating aerobic and anaerobic ammonium-oxidizing bacteria (AerAOB and AnAOB). The goal of this study was to verify putative mutual benefits for aggregated AerAOB and AnAOB in a stagnant freshwater environment. In an ammonium fed water column, the biological oxygen consumption rate was, on average, 76 kg O₂ ha⁻¹ day⁻¹. As the oxygen transfer rate of an abiotic control column was only 17 kg O₂ ha⁻¹ day⁻¹, biomass activity enhanced the oxygen transfer. Increasing the AnAOB gas production increased the oxygen consumption rate with more than 50% as a result of enhanced vertical movement of the biomass. The coupled decrease in dissolved oxygen concentration increased the diffusional oxygen transfer from the atmosphere in the water. Physically preventing the biomass from rising to the upper water layer instantaneously decreased oxygen and ammonium consumption and even led to the occurrence of some sulfate reduction. Floating of the biomass was further confirmed to be beneficial, as this allowed for the development of a higher AerAOB and AnAOB activity, compared to settled biomass. Overall, the results support mutual benefits for aggregated AerAOB and AnAOB, derived from the biomass uplifting effect of AnAOB gas production.

Keywords: OLAND; Nitrification; Nitritation; Anammox; Oxygen transfer; Lagoon

Co-existence of physiologically similar sulfate-reducing bacteria in a full-scale sulfidogenic bioreactor fed with a single organic electron donor by Shabir A. Dar; Alfons J. M. Stams; J. Gijs Kuenen; Gerard Muyzer (pp. 1463-1472).

A combination of culture-dependent and independent methods was used to study the co-existence of different sulfate-reducing bacteria (SRB) in an upflow anaerobic sludge bed reactor treating sulfate-rich wastewater. The wastewater was fed with ethanol as an external electron donor. Twenty six strains of SRB were randomly picked and isolated from the highest serial dilution that showed growth (i.e. 10⁸). Repetitive enterobacterial palindromic polymerase chain reaction and whole cell protein profiling revealed a low genetic diversity, with only two genotypes among the 26 strains obtained in the pure culture. The low genetic diversity suggests the absence of micro-niches within the reactor, which might be due to a low spatial and temporal micro-heterogeneity. The total 16S rDNA sequencing of two representative strains L3 and L7 indicated a close relatedness to the genus Desulfovibrio. The two strains differed in as many as five physiological traits, which might allow them to occupy distinct niches and thus co-exist within the same habitat. Whole cell hybridisation with fluorescently labeled oligonucleotide probes was performed to characterise the SRB community in the reactor. The isolated strains Desulfovibrio L3 and Desulfovibrio L7 were the most dominant SRB, representing 30–35% and 25–35%, respectively, of the total SRB community. Desulfobulbus-like bacteria contributed for 20–25%, and the Desulfobacca acetoxidans-specific probe targeted approximately 15–20% of the total SRB. The whole cell hybridisation results thus revealed a consortium of four different species of SRB that can be enriched and maintained on a single energy source in a full-scale sulfidogenic reactor.

Keywords: Sulfate-reducing bacteria; Microbial ecology; Ecological niches

Spectrophotometric assay for detection of aromatic hydroxylation catalyzed by fungal haloperoxidase–peroxygenase by Martin G. Kluge; René Ullrich; Katrin Scheibner; Martin Hofrichter (pp. 1473-1478).

Agrocybe aegerita peroxidase (AaP) is a versatile heme-thiolate protein that can act as a peroxygenase and catalyzes, among other reactions, the hydroxylation of aromatic rings. This paper reports a rapid and selective spectrophotometric method for directly detecting aromatic hydroxylation by AaP. The weakly activated aromatic compound naphthalene served as the substrate that was regioselectively converted into 1-naphthol in the presence of the co-substrate hydrogen peroxide. Formation of 1-naphthol was followed at 303 nm (ɛ ₃₀₃ = 2,010 M⁻¹ cm⁻¹), and the apparent Michaelis–Menten (K _m) and catalytic (k _cat) constants for the reaction were estimated to be 320 μM and 166 s⁻¹, respectively. This method will be useful in screening of fungi and other microorganisms for extracellular peroxygenase activities and in comparing and assessing different catalytic activities of haloperoxidase–peroxygenases.

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Applied Microbiology and Biotechnology (v.75, #6)

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Applied Microbiology and Biotechnology (v.75, #6)