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Applied Microbiology and Biotechnology (v.65, #1)

Tissue engineering: current state and perspectives by E. Lavik; R. Langer (pp. 1-8).

Tissue engineering is an interdisciplinary field that involves cell biology, materials science, reactor engineering, and clinical research with the goal of creating new tissues and organs. Significant advances in tissue engineering have been made through improving singular aspects within the overall approach, e.g., materials design, reactor design, or cell source. Increasingly, however, advances are being made by combining several areas to create environments which promote the development of new tissues whose properties more closely match their native counterparts. This approach does not seek to reproduce all the complexities involved in development, but rather seeks to promote an environment which permits the native capacity of cells to integrate, differentiate, and develop new tissues. Progenitors and stem cells will play a critical role in understanding and developing new engineered tissues as part of this approach.

A survey of computational and physical methods applied to solid-state fermentation by J. Lenz; M. Höfer; J.-B. Krasenbrink; U. Hölker (pp. 9-17).

During the last decade, significant effort has been made to apply computational and physical methods to solid-state fermentation (SSF). This had positive impact both on our understanding of the basic principles underlying this old technology, and on the latest progress made in industrial bioengineering. Guidelines on bioreactor design and operation including scale-up, new methods for biomonitoring and advanced control strategies are among the most important outcomes of practical use. Nevertheless, there still is a lack of experimental data, which hampers parameter identification and thus broader use of mathematical modeling. More attention should therefore be paid to combining and concentrating modern physical techniques and computational approaches in order to allow better model validation and thus further progress in rational bioengineering of SSF.

Monitoring the progress of infection and recombinant protein production in insect cell cultures using intracellular ATP measurement by A. M. Olejnik; K. Czaczyk; R. Marecik; W. Grajek; T. Jankowski (pp. 18-24).

Several monitoring methods used to predict viable cell density have been the subject of extensive studies, including oxygen uptake rate, carbon dioxide evolution rate, optical density, NADH-dependent fluorescence and relative permittivity measurement . We propose intracellular ATP determination by bioluminescence assay to monitor the progress of baculovirus infection and recombinant protein production in insect cell cultures. We found that the ATP content in viable cells increased after virus addition. The increase in the ATP level was observed until the maximum recombinant protein accumulation was reached. At maximum product yield, the specific ATP content significantly decreased. Results obtained in both batch and fed-batch cultures demonstrated that the specific ATP level could be considered as a good indicator of recombinant protein productivity. Monitoring the cellular ATP content after viral infection makes it possible to define the optimum time for product harvest. The main advantage of applying the ATP assay as an index of the progress of infection and recombinant protein synthesis is its short time and sensitivity.

Assimilation of grape phytosterols by Saccharomyces cerevisiae and their impact on enological fermentations by V. Luparia; V. Soubeyrand; T. Berges; A. Julien; J.-M. Salmon (pp. 25-32).

Although yeasts are known to be able to incorporate a wide variety of exogenous sterols under strict anaerobiosis, no data are available on the assimilation of grapevine phytosterols under enological conditions and the eventual impact on fermentation kinetics. We used therefore a mixture of pure phytosterols, in a proportion representative of the different grape skins phytosterols, to supplement a synthetic fermentation medium simulating a grape must. Under anaerobiosis, normal biomass formation was achieved with 5 mg phytosterols l⁻¹. Similar results were obtained in comparison with the observed maximal fermentation rates. These results clearly indicated that grape phytosterols may efficiently act as a substitute for ergosterol in the yeast membrane for promoting yeast growth and initial fermentative activity. Analysis of total yeast sterols indicated that phytosterols are accumulated without further modification, mainly in their esterified form. However, all the fermentations performed with synthetic media supplemented with phytosterols led to stuck fermentations, linked to a correlative strong decrease in cell viability during the stationary phase. Therefore, grape phytosterols are easily incorporated by yeast cells under enological conditions for promoting initial growth and fermentative activity, but rapidly perturb the yeast membrane properties by being the predominant sterols.

Steady-state and transient-state performance of a biotrickling filter treating chlorobenzene-containing waste gas by C. Seignez; N. Adler; C. Thoeni; M. Stettler; P. Péringer; C. Holliger (pp. 33-37).

Biotrickling filter (BTF) technology was applied for the treatment of waste gas containing a mixture of chlorobenzene and 1,2-dichlorobenzene. An adapted microbial community was immobilised on a structured packing material. The strategy followed was to reach high removal efficiencies at initially low mass loading rates followed by an increase of the latter. This procedure was successful and resulted in a short start-up period of only 2 weeks. A 3-month operation under steady-state conditions showed good performance, with >95% removal efficiency at a mass loading rate of 1,800 g m⁻³ day⁻¹. Dimensionless concentration profiles showed that the chlorobenzenes were simultaneously degraded. Low dissolved organic carbon of 15 mg l⁻¹ and stoichiometric chloride concentrations in the trickling liquid indicated complete mineralisation of the pollutant. Transient-state experiments with five times higher mass loading rates caused a decrease in the removal efficiency that recovered rapidly once the mass loading rate returned to its original steady-state level. A progressive increase of the mass loading rate in a long-term performance experiment showed that the removal efficiency could be kept stable between 95 and 99% at loads of up to 5,200 g m⁻³ day⁻¹ over several days. Above this mass loading rate, the elimination capacity did not increase any further. These results demonstrated that with a well-adapted inoculum and optimal operation parameters, a BTF system with excellent performance and stability that efficiently removes a mixture of cholorobenzene vapours from air can be obtained.

Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila by Ksenia Egorova; Harald Trauthwein; Stefan Verseck; Garabed Antranikian (pp. 38-45).

A constitutively expressed thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila was purified to homogeneity by applying hydrophobic interaction, anion exchange and gel filtration chromatography, giving a yield of 26% and a specific activity of 19.5 units mg⁻¹. The purified enzyme has an estimated molecular mass of 108 kDa and an isoelectric point of 4.2. The amidase is active at a broad pH range (pH 4–9) and temperature range (40–80°C) and has a half-life of 1.2 h at 70°C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co²⁺, Hg²⁺, Cu²⁺, Ni²⁺, and thiol reagents. The amidase has a broad substrate spectrum, including aliphatic, aromatic and amino acid amides. The presence of a double bond or a methyl group near the carboxamide group of aliphatic and amino acid amides enhances the enzymatic activity. Among aromatic amides with substitutions at the o-, m-, or p-position, the p-substituted amides are the preferred substrates. The highest acyl transferase activity was detected with hexanoamide, isobutyramide and propionamide. The K _m values for propionamide, methacrylamide, benzamide and 2-phenylpropionamide are 7.4, 9.2, 4.9 and 0.9 mM, respectively. The amidase is highly S-stereoselective for 2-phenylpropionamide; and the racemic amide was converted to the corresponding S-acid with an enantiomeric excess of >95% at 50% conversion of the substrate. In contrast, the d,l-tryptophanamide and d,l-methioninamide were converted to the corresponding d,l-acids at the same rate. This thermostable enzyme represents the first reported amidase from a thermophilic actinomycete.

Isolation and characterization of PRA1, a trypsin-like protease from the biocontrol agent Trichoderma harzianum CECT 2413 displaying nematicidal activity by Belen Suarez; Manuel Rey; Pablo Castillo; Enrique Monte; Antonio Llobell (pp. 46-55).

Mycoparasitic Trichoderma strains secrete a complex set of hydrolytic enzymes under conditions related to antagonism. Several proteins with proteolytic activity were detected in culture filtrates from T. harzianum CECT 2413 grown in fungal cell walls or chitin and the protein responsible for the main activity (PRA1) was purified to homogeneity. The enzyme was monomeric, its estimated molecular mass was 28 kDa (SDS-PAGE), and its isoelectric point 4.7–4.9. The substrate specificity and inhibition profile of the enzyme correspond to a serine-protease with trypsin activity. Synthetic oligonucleotide primers based on N-terminal and internal sequences of the protein were designed to clone a full cDNA corresponding to PRA1. The protein sequence showed <43% identity to mammal trypsins and 47–57% to other fungal trypsin-like proteins described thus far. Northern analysis indicated that PRA1 is induced by conditions simulating antagonism, is subject to nitrogen and carbon derepression, and is affected by pH in the culture media. The number of hatched eggs of the root-knot nematode Meloidogyne incognita was significantly reduced after incubation with pure PRA1 preparations. This nematicidal effect was improved using fungal culture filtrates, suggesting that PRA1 has additive or synergistic effects with other proteins produced during the antagonistic activity of T. harzianum CECT 2413. A role for PRA1 in the protection of plants against pests and pathogens provided by T. harzianum CECT 2413 is proposed.

Aerobic production of alanine by Escherichia coli aceF ldhA mutants expressing the Bacillus sphaericus alaD gene by M. Lee; G. M. Smith; M. A. Eiteman; E. Altman (pp. 56-60).

Alanine was produced from glucose in an Escherichia coli aceF ldhA double mutant strain that contained the pTrc99A-alaD plasmid expressing Bacillus sphaericus alanine dehydrogenase. The aceF gene encodes one of the proteins of the pyruvate dehydrogenase complex, and therefore this strain required acetate as an additional carbon source. The ldhA gene encodes fermentative lactate dehydrogenase, a competitor of alanine dehydrogenase for the substrate pyruvate. Fermentations included an oxygenated growth phase followed by an oxygen-limited alanine production phase. The lowest value for the mass transfer coefficient (k _La) studied during the production phase yielded the greatest alanine. With feeding of glucose and NH₄Cl, 32 g/l alanine accumulated in 27 h with a yield of 0.63 g alanine generated per gram glucose consumed.

A food-grade delivery system for Lactococcus lactis and evaluation of inducible gene expression by A. Simões-Barbosa; H. Abreu; A. Silva Neto; A. Gruss; P. Langella (pp. 61-67).

The genetic improvement of Lactococcus lactis is a matter of biotechnological interest in the food industry and in the pharmaceutical and medical fields. However, to construct a food-grade delivery system, both the presence of antibiotic markers or plasmid sequences should be avoided and the maintenance and expression of the cloned gene should be guaranteed. The objective of this work was to produce crossover mutants of L. lactis with a reporter gene under the control of an inducible promoter in order to evaluate the level of gene expression. We utilized a nuclease gene of Staphylococcus aureus as a reporter gene, P nisA as the nisin-inducible promoter, a non-essential gene involved in histidine biosynthesis of L. lactis as the site for homologous recombination, and pRV300 as a suicide vector for the genomic integration in L. lactis NZ9000. Single- and double-crossover mutants were identified by genotype and phenotype. Relative to episomal transformants of L. lactis, the level of expression of the heterologous protein after nisin induction was similar in the crossover mutants, suggesting that a single copy of the heterologous gene can be used to produce the protein of interest.

Construction of a self-cloning sake yeast that overexpresses alcohol acetyltransferase gene by a two-step gene replacement protocol by I. Hirosawa; K. Aritomi; H. Hoshida; S. Kashiwagi; Y. Nishizawa; R. Akada (pp. 68-73).

The commercial application of genetically modified industrial microorganisms has been problematic due to public concerns. We constructed a “self-cloning” sake yeast strain that overexpresses the ATF1 gene encoding alcohol acetyltransferase, to improve the flavor profile of Japanese sake. A constitutive yeast overexpression promoter, TDH3p, derived from the glyceraldehyde-3-phosphate dehydrogenase gene from sake yeast was fused to ATF1; and the 5′ upstream non-coding sequence of ATF1 was further fused to TDH3p-ATF1. The fragment was placed on a binary vector, pGG119, containing a drug-resistance marker for transformation and a counter-selection marker for excision of unwanted DNA. The plasmid was integrated into the ATF1 locus of a sake yeast strain. This integration constructed tandem repeats of ATF1 and TDH3p-ATF1 sequences, between which the plasmid was inserted. Loss of the plasmid, which occurs through homologous recombination between either the TDH3p downstream ATF1 repeats or the TDH3p upstream repeat sequences, was selected by growing transformants on counter-selective medium. Recombination between the downstream repeats led to reversion to a wild type strain, but that between the upstream repeats resulted in a strain that possessed TDH3p-ATF1 without the extraneous DNA sequences. The self-cloning TDH3p-ATF1 yeast strain produced a higher amount of isoamyl acetate. This is the first expression-controlled self-cloning industrial yeast.

Transcriptional analysis of genes for energy catabolism and hydrolytic enzymes in the filamentous fungus Aspergillus oryzae using cDNA microarrays and expressed sequence tags by Hiroshi Maeda; Motoaki Sano; Yutaka Maruyama; Takeki Tanno; Takeshi Akao; Yoshiteru Totsuka; Misako Endo; Rumi Sakurada; Youhei Yamagata; Masayuki Machida; Osamu Akita; Fumihiko Hasegawa; Keietsu Abe; Katsuya Gomi; Tasuku Nakajima; Yasutaka Iguchi (pp. 74-83).

Aspergillus oryzae is a fungus used extensively in the fermentation industry. We constructed cDNA microarrays comprising 2,070 highly expressed cDNAs selected from the ∼6,000 non-redundant expressed sequence tags (ESTs) in the A. oryzae EST database ( http://www.aist.go.jp/RIODB/ffdb/index.html ). Using the cDNA microarrays, we analyzed the gene expression profiles of A. oryzae cells grown under the glucose-rich (AC) and glucose-depleted (AN) liquid culture conditions used during the construction of the EST database. The sets of genes identified by the cDNA microarray as highly expressed under each culture condition agreed well with the highly redundant ESTs obtained under the same conditions. In particular, transcription levels of most catabolic genes of the glycolytic pathway (EMP) and tricarboxylic acid (TCA) cycle were higher under AC than AN conditions, suggesting that A. oryzae uses both EMP and TCA for glucose metabolism under AC conditions. We further studied the expression of genes encoding hydrolytic enzymes and enzymes involved in energy catabolism by using three industrial solid-phase biomass media, including wheat-bran. The wheat-bran culture gave the richest gene expression profile of hydrolytic enzymes and the lowest expression levels of catabolic genes (EMP, TCA) among the three media tested. The low expression levels of catabolic genes in the wheat-bran culture may release catabolite repression, consequently leading to the rich expression profiles of the hydrolytic enzymes.

Metabolic regulation analysis of icd-gene knockout Escherichia coli based on 2D electrophoresis with MALDI-TOF mass spectrometry and enzyme activity measurements by M. Mohiuddin Kabir; Kazuyuki Shimizu (pp. 84-96).

An integrated study of cell growth characteristics, enzyme activities and protein expression patterns was carried out to investigate how the central metabolism of Escherichia coli changes upon knockout of the isocitrate dehydrogenase (ICDH) gene (icd) in the tricarboxylic acid cycle. Deletion of the icd gene led to reduced specific growth rate and reduced specific glucose consumption rate. The reduced specific growth rate in the icd mutant was due mainly to the lower intracellular ATP/ADP ratio as well as to the lower NADPH/NADP⁺ ratio compared with those in the parent strain. However, the specific carbon dioxide evolution rate was found to be higher in the icd mutant strain compared to the parent E. coli. This may be due to the higher activity of 6-phosphogluconate dehydrogenase, phosphoenol pyruvate carboxykinase and NADP⁺-dependent malic enzymes. The glyoxylate pathway was also utilized, as evidenced by the significant upregulation of isocitrate lyase and malate synthase activity in the icd mutant E. coli. The appearance of the glyoxylate pathway caused lower acetate production. Of 21 proteins showing altered expression levels, 17 were successfully identified with the aid of MALDI-TOF mass spectrometry. The results showed that the abolition of ICDH activity significantly affected the respiratory system and electron transport chain, as evidenced by the significant downregulation of proteins encoded by the genes nuoE, nuoH, cydA and cyoA in icd mutant E. coli compared to the parent.

Colonization, biofilm formation and biodegradation of polyethylene by a strain of Rhodococcus ruber by I. Gilan (Orr); Y. Hadar; A. Sivan (pp. 97-104).

A two-step enrichment procedure led to the isolation of a strain of Rhodococcus ruber (C208) that utilized polyethylene films as sole carbon source. In liquid culture, C208 formed a biofilm on the polyethylene surface and degraded up to 8% (gravimetrically) of the polyolefin within 30 days of incubation. The bacterial adhesion to hydrocarbon assay and the salt aggregation test both showed that the cell-surface hydrophobicity of C208 was higher than that of three other isolates which were obtained from the same consortium but were less efficient than C208 in the degradation of polyethylene. Mineral oil, but not nonionic surfactants, enhanced the colonization of polyethylene and increased biodegradation by about 50%. Fluorescein diacetate (FDA) hydrolysis and protein content analysis were used to test the viability and biomass density of the C208 biofilm on the polyethylene, respectively. Both FDA activity and protein content of the biofilm in a medium containing mineral oil peaked 48–72 h after inoculation and then decreased sharply. This finding apparently reflected rapid utilization of the mineral oil adhering to the polyethylene. The remaining biofilm population continued to proliferate moderately and presumably played a major role in biodegradation of the polyethylene. Fourier transform infrared spectra of UV-photooxidized polyethylene incubated with C208 indicated that biodegradation was initiated by utilization of the carbonyl residues formed in the photooxidized polyethylene

Bacillus amyloliquefaciens phage endolysin can enhance permeability of Pseudomonas aeruginosa outer membrane and induce cell lysis by Y. Orito; M. Morita; K. Hori; H. Unno; Y. Tanji (pp. 105-109).

To determine the function of the C-terminal region of Bacillus amyloliquefaciens phage endolysin on Pseudomonas aeruginosa lysis, the permeabilization of the outer membrane of P. aeruginosa was analyzed. Glu-15 to His (E15H) and Thr-32 to Glu (T32E) substitutions were introduced into the Bacillus phage endolysin. Neither E15H nor T32E substitution induced enzymatic and antibacterial activities. These two, Glu-15 and Thr-32, were considered to be the active center of the enzyme. The addition of purified E15H and T32E proteins to P. aeruginosa cells induced the release of periplasmic β-lactamase from the cells, indicating that both proteins enhance permeabilization of the outer membrane. However, the addition of E15H and T32E proteins to P. aeruginosa cells did not induce the release of cytoplasmic ATP from the cells. These results indicate that the antibacterial activity of the endolysin requires both the C-terminal enhancement of the permeabilization of the P. aeruginosa outer membrane and N-terminal enzymatic activity.

Molecular and physiological characterisation of a 3-phytase from soil bacterium Klebsiella sp. ASR1 by A. Sajidan; A. Farouk; R. Greiner; P. Jungblut; E.-C. Müller; R. Borriss (pp. 110-118).

Klebsiella sp. strain ASR1 isolated from an Indonesian rice field is able to hydrolyse myo-inositol hexakis phosphate (phytate). The phytase protein was purified and characterised as a 42 kDa protein accepting phytate, NADP and sugar phosphates as substrates. The corresponding gene (phyK) was cloned from chromosomal DNA using a combined approach of protein and genome analysis, and expressed in Escherichia coli. The recombinant enzyme was identified as a 3-phytase yielding myo-inositol monophosphate, Ins(2)P, as the final product of enzymatic phytate hydrolysis. Based on its amino acid sequence, PhyK appears to be a member of a hitherto unknown subfamily of histidine acid phytate-degrading enzymes with the active site RHGXRXP and HD sequence motifs, and is different from other general phosphatases and phytases. Due to its ability to degrade sodium phytate to the mono phosphate ester, the phyK gene product is an interesting candidate for industrial and agricultural applications to make phytate phosphorous available for plant and animal nutrition.

Effect of pH on Lactobacillus fermentum growth, raffinose removal, α-galactosidase activity and fermentation products by J. G. LeBlanc; M. S. Garro; G. Savoy de Giori (pp. 119-123).

In this study, the behaviour of Lactobacillus fermentum CRL 722 and CRL 251 were evaluated under different pH conditions (pH 6.0, 5.5, 5.0, 4.5) and without pH control. Growth was similar under all conditions assayed except at pH 4.5. These microorganisms were able to eliminate raffinose, a nondigestible α-oligosaccharide (NDO) found in soy products, showing a consumption rate of 0.25 g l⁻¹ h⁻¹ (pH 6.0–5.0). The removal of raffinose was due to the high α-galactosidase (α-gal) activities of these lactic acid bacteria, which was highest at pH 5.5 (5.0 U/ml). The yield of organic acids produced during raffinose consumption was also highest at this pH. The results of this study will allow selection of the optimum growth conditions of L. fermentum with elevated levels of α-gal to be used in the reduction of NDO in soy products when used as starter cultures.

Isolation and characterization of novel bacteria degrading polycyclic aromatic hydrocarbons from polluted Greek soils by Haimou Zhang; Aristeidis Kallimanis; Anna I. Koukkou; Constantin Drainas (pp. 124-131).

Three bacterial strains, designated as Wphe1, Sphe1, and Ophe1, were isolated from Greek soils contaminated with polycyclic aromatic hydrocarbon (PAH)-containing waste from the wood processing, steel, and oil refinery industries. Wphe1, Sphe1, and Ophe1 were characterized and identified as species of Pseudomonas, Microbacterium, and Paracoccus, respectively, based on Gram staining, biochemical tests, phospholipid analysis, FAME analysis, G+C content and 16S rRNA gene sequence analysis. The results of gas chromatography showed that strain Wphe1 degraded naphthalene, phenanthrene, and m-cresol over a wide temperature range; strain Sphe1 was a degrader of phenanthrene and n-alkanes; most interestingly, strain Ophe1 degraded anthracene, phenanthrene, fluorene, fluoranthene, chrysene, and pyrene, as well as cresol compounds and n-alkanes as sole carbon source. This is the first report of a representative of the genus Paracoccus capable of degrading PAHs with such versatility. These three strains may be useful for bioremediation applications.

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Applied Microbiology and Biotechnology (v.65, #1)