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Applied Microbiology and Biotechnology (v.82, #6)

Biosynthetic pathways for 3-hydroxypropionic acid production by Xinglin Jiang; Xin Meng; Mo Xian (pp. 995-1003).

Biobased platform chemicals have attracted growing interest recently. Among them, 3-hydroxypropionic acid receives significant attention due to its applications in the synthesis of novel polymer materials and other derivatives. To establish a biotechnology route instead of the problematic chemical synthesis of 3-hydroxypropionic acid, biosynthetic pathway is required, and the strategies of how to engineer a microbe to produce this product should be considered. In the present review, we summarize and review all known pathways, which could be potentially constructed for 3-hydroxypropionic acid production. Mass and redox balances are discussed in detail. Thermodynamic favorability is evaluated by standard Gibbs free energy. The assembly of pathways and possible solutions are proposed. Several new techniques and future research needs are also covered.

Keywords: 3-Hydroxypropionic acid; Pathway; Fermentation; Thermodynamics

Microbial and enzymatic control of pitch in the pulp and paper industry by Ana Gutiérrez; José C. del Río; Angel T. Martínez (pp. 1005-1018).

Pitch control is an important aspect in pulp and paper manufacture, and the first example where microbial biotechnology provided successful solutions in this industrial sector. Triglycerides cause deposits in softwood mechanical pulping, and both microbial and enzymatic products have been commercialized to be applied on wood and pulp, respectively. The former are based on colorless strains of sapstain fungi. The latter are improved lipases, including thermostable variants from directed evolution. These enzymes are among the additives of choice in pulping of high-resin-content softwoods. However, lipases are not useful when pitch originates from other lipids, such as steroids and terpenes, and the sapstain inocula are also only partially effective. In the search for stronger biocatalysts to degrade recalcitrant lipids, the potential of white-rot fungi and their enzymes has been demonstrated. When inocula of these fungi are used, wood treatment must be controlled to avoid cellulose degradation. However, the efficiency and selectivity of the laccase-mediator system permits its integration as an additional bleaching stage. A double benefit can be obtained from these treatments since pitch is controlled at the same time that residual lignin is removed facilitating the implementation of totally chlorine free pulp bleaching.

Keywords: Pitch deposits; Paper pulps; Wood fungi; Fungal enzymes; Laccase-mediator system

Bioindicator production with Bacillus atrophaeus’ thermal-resistant spores cultivated by solid-state fermentation by Sandra Regina B. R. Sella; Belquis P. Guizelini; Luciana P. S. Vandenberghe; Adriane B. P. Medeiros; Carlos Ricardo Soccol (pp. 1019-1026).

Bacillus atrophaeus’ spores are used in the preparation of bioindicators to monitor the dry heat, ethylene oxide, and plasma sterilization processes and in tests to assess sterilizing products. Earlier production methods involved culture in chemically defined medium to support sporulation with the disadvantage of requiring an extended period of time (14 days) besides high cost of substrates. The effect of cultivation conditions by solid-state fermentation (SSF) was investigated aiming at improving the cost–productivity relation. Initial SSF parameters such as the type of substrate were tested. Process optimization was carried out using factorial experimental designs and response surface methodology in which the influence of different variables—particle size, moisture content, incubation time, pH, inoculum size, calcium sources, and medium composition—was studied. The results have suggested that soybean molasses and sugarcane bagasse are potential substrate and support, respectively, contributing to a 5-day reduction in incubation time. Variables which presented significant effects and optimum values were mean particle size (1.0 mm), moisture content (93%), initial substrate pH (8.0), and water as a solution base. The high-yield spore production was about 3 logs higher than the control and no significant difference in dry heat resistance was observed.

Keywords: Bacillus atrophaeus ; Bioindicator; Solid-state fermentation; Sugar cane bagasse; Response surface methodology; Optimization

Net effect of wort osmotic pressure on fermentation course, yeast vitality, beer flavor, and haze by K. Sigler; D. Matoulková; M. Dienstbier; P. Gabriel (pp. 1027-1035).

The net effect of increased wort osmolarity on fermentation time, bottom yeast vitality and sedimentation, beer flavor compounds, and haze was determined in fermentations with 12° all-malt wort supplemented with sorbitol to reach osmolarity equal to 16° and 20°. Three pitchings were performed in 12°/12°/12°, 16°/16°/12°, and 20°/20°/12° worts. Fermentations in 16° and 20° worts decreased yeast vitality measured as acidification power (AP) by a maximum of 10%, lowered yeast proliferation, and increased fermentation time. Repitching aggravated these effects. The 3rd “back to normal” pitching into 12° wort restored the yeast AP and reproductive abilities while the extended fermentation time remained. Yeast sedimentation in 16° and 20° worts was delayed but increased about two times at fermentation end relative to that in 12° wort. Third “back-to-normal” pitching abolished the delay in sedimentation and reduced its extent, which became nearly equal in all variants. Beer brewed at increased osmolarity was characterized by increased levels of diacetyl and pentanedione and lower levels of dimethylsulfide and acetaldehyde. Esters and higher alcohols displayed small variations irrespective of wort osmolarity or repitching. Increased wort osmolarity had no appreciable effect on the haze of green beer and accelerated beer clarification during maturation. In all variants, chill haze increased with repitching.

Keywords: Wort osmolarity; High-gravity brewing; Fermentation course; Yeast vitality; Flavor compounds; Beer haze

Alcoholic fermentation of xylose and mixed sugars using recombinant Saccharomyces cerevisiae engineered for xylose utilization by Anjali Madhavan; Sriappareddy Tamalampudi; Aradhana Srivastava; Hideki Fukuda; Virendra S. Bisaria; Akihiko Kondo (pp. 1037-1047).

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.

Keywords: Orpinomyces xylose isomerase; Recombinant Saccharomyces cerevisiae ; Xylose fermentation; Ethanol; Adaptation; Borate

Continuous 2-keto-l-gulonic acid fermentation from l-sorbose by Ketogulonigenium vulgare DSM 4025 by Yoshinori Takagi; Teruhide Sugisawa; Tatsuo Hoshino (pp. 1049-1056).

A single-stage continuous fermentation process for the production of 2-keto-l-gulonic acid (2KGA) from l-sorbose using Ketogulonigenium vulgare DSM 4025 was developed. The chemostat culture with the dilution rate that was calculated based on the relationship between the 2KGA production rate and the 2KGA concentration was feasible for production with high concentration of 2KGA. In this system, 112.2 g/L of 2KGA on the average was continuously produced from 114 g/L of l-sorbose. A steady state of the fermentation was maintained for the duration of more than 110 h. The dilution rate was kept in the range of 0.035 and 0.043 h⁻¹, and the 2KGA productivity was 3.90 to 4.80 g/L/h. The average molar conversion yield of 2KGA from l-sorbose was 91.3%. Under the optimal conditions, l-sorbose concentration was kept at 0 g/L. Meanwhile, the dissolved oxygen level was changing in response to the dilution rate and 2KGA concentration. In the dissolved oxygen (DO) range of 16% to 58%, it was revealed that the relationship between DO and D possessed high degree of positive correlation under the l-sorbose limiting condition (complete consumption of l-sorbose). Increasing D closer to the critical value for washing out point of the continuous fermentation, DO value tended to be gradually increased up to 58%. In conclusion, an efficient and reproducible continuous fermentation process for 2KGA production by K. vulgare DSM 4025 could be developed using a medium containing baker’s yeast without using a second helper microorganism.

Keywords: 2-Keto-l-gulonic acid; l-Sorbose; Continuous fermentation; Ketogulonicigenium vulgare

Conversion of dibenzothiophene by the mushrooms Agrocybe aegerita and Coprinellus radians and their extracellular peroxygenases by Elizabet Aranda; Matthias Kinne; Martin Kluge; René Ullrich; Martin Hofrichter (pp. 1057-1066).

The conversion of the heterocycle dibenzothiophene (DBT) by the agaric basidiomycetes Agrocybe aegerita and Coprinellus radians was studied in vivo and in vitro with whole cells and with purified extracellular peroxygenases, respectively. A. aegerita oxidized DBT (110 μM) by 100% within 16 days into eight different metabolites. Among the latter were mainly S-oxidation products (DBT sulfoxide, DBT sulfone) and in lower amounts, ring-hydroxylation compounds (e.g., 2-hydroxy-DBT). C. radians converted about 60% of DBT into DBT sulfoxide and DBT sulfone as the sole metabolites. In vitro tests with purified peroxygenases were performed to compare the product pattern with the metabolites formed in vivo. Using ascorbic acid as radical scavenger, a total of 19 and seven oxygenation products were detected after DBT conversion by the peroxygenases of A. aegerita (AaP) and C. radians (CrP), respectively. Whereas ring hydroxylation was favored over S-oxidation by AaP (again 2-hydroxy-DBT was identified), CrP formed DBT sulfoxide as major product. This finding suggests that fungal peroxygenases can considerably differ in their catalytic properties. Using H₂ ¹⁸O₂, the origin of oxygen was proved to be the peroxide. Based on these results, we propose that extracellular peroxygenases may be involved in the oxidation of heterocycles by fungi also under natural conditions.

Keywords: Basidiomycete; Peroxygenase; Peroxidase; Dibenzothiophene; Sulfoxidation; Hydroxylation

Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol by Anjali Madhavan; Sriappareddy Tamalampudi; Kazunari Ushida; Daisuke Kanai; Satoshi Katahira; Aradhana Srivastava; Hideki Fukuda; Virendra S. Bisaria; Akihiko Kondo (pp. 1067-1078).

The cDNA sequence of the gene for xylose isomerase from the rumen fungus Orpinomyces was elucidated by rapid amplification of cDNA ends. The 1,314-nucleotide gene was cloned and expressed constitutively in Saccharomyces cerevisiae. The deduced polypeptide sequence encoded a protein of 437 amino acids which showed the highest similarity to the family II xylose isomerases. Further, characterization revealed that the recombinant enzyme was a homodimer with a subunit of molecular mass 49 kDa. Cell extract of the recombinant strain exhibited high specific xylose isomerase activity. The pH optimum of the enzyme was 7.5, while the low temperature optimum at 37°C was the property that differed significantly from the majority of the reported thermophilic xylose isomerases. In addition to the xylose isomerase gene, the overexpression of the S. cerevisiae endogenous xylulokinase gene and the Pichia stipitis SUT1 gene for sugar transporter in the recombinant yeast facilitated the efficient production of ethanol from xylose.

Keywords: Xylose isomerase; Orpinomyces ; Xylulokinase; SUT1 ; Recombinant Saccharomyces cerevisiae ; Xylose fermentation; Ethanol

Comparative analysis of four β-galactosidases from Bifidobacterium bifidum NCIMB41171: purification and biochemical characterisation by Theodoros Goulas; Athanasios Goulas; George Tzortzis; Glenn R. Gibson (pp. 1079-1088).

Four different β-galactosidases (previously named BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171 were overexpressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. BbgI was forming a hexameric protein complex of 875 kDa, whereas BbgII, BbgIII and BbgIV were dimers with native molecular masses of 178, 351 and 248 kDa, respectively. BbgII was the only enzyme that preferred acidic conditions for optimal activity (pH 5.4–5.8), whereas the other three exhibited optima in more neutral pH ranges (pH 6.4–6.8). Na⁺ and/or K⁺ ions were prerequisite for BbgI and BbgIV activity in Bis–Tris-buffered solutions, whereas Mg⁺⁺ was strongly activating them in phosphate-buffered solutions. BbgII and BbgIII were slightly influenced from the presence or absence of cations, with Mg⁺⁺, Mn⁺⁺ and Ca⁺⁺ ions exerting the most positive effect. Determination of the specificity constants (k _cat/K _m) clearly indicated that BbgI (6.11 × 10⁴ s⁻¹ M⁻¹), BbgIII (2.36 × 10⁴ s⁻¹ M⁻¹) and especially BbgIV (4.01 × 10⁵ s⁻¹ M⁻¹) are highly specialised in the hydrolysis of lactose, whereas BbgII is more specific for β-d-(1→6) galactobiose (5.59 × 10⁴ s⁻¹ M⁻¹) than lactose (1.48 × 10³ s⁻¹ M⁻¹). Activity measurements towards other substrates (e.g. β-d-(1→6) galactobiose, β-d-(1→4) galactobiose, β-d-(1→4) galactosyllactose, N-acetyllactosamine, etc.) indicated that the β-galactosidases were complementary to each other by hydrolysing different substrates and thus contributing in a different way to the bacterial physiology.

Keywords: Bifidobacterium bifidum ; Bifidobacteria; Galactosidase; Prebiotic

Characterization of a regulatory gene, aveR, for the biosynthesis of avermectin in Streptomyces avermitilis by Shigeru Kitani; Haruo Ikeda; Takako Sakamoto; Satoru Noguchi; Takuya Nihira (pp. 1089-1096).

Avermectin is an important macrocyclic polyketide produced by Streptomyces avermitilis and widely used as an anthelmintic agent in the medical, veterinary, and agricultural fields. The avermectin biosynthetic gene cluster contains aveR, which belongs to the LAL-family of regulatory genes. In this study, aveR was inactivated by gene replacement in the chromosome of S. avermitilis, resulting in the complete loss of avermectin production. The aveR mutant was unable to convert an avermectin intermediate to any avermectin derivatives, and complementation by intact aveR and its proper upstream region restored avermectin production in the mutant, suggesting that AveR is a positive regulator controlling the expression of both polyketide biosynthetic genes and postpolyketide modification genes in avermectin biosynthesis. Despite the general concept that an increased amount of a positive pathway-specific regulator leads to higher production, a higher amount of aveR resulted in complete loss of avermectin, indicating that there is a maximum threshold concentration of aveR for the production of avermectin.

Keywords: Avermectin; Regulatory gene; LAL-family transcriptional regulator

Design and characterization of novel hybrid peptides from LFB15(W4,10), HP(2-20), and cecropin A based on structure parameters by computer-aided method by Zi-gang Tian; Tian-tang Dong; Da Teng; Ya-lin Yang; Jian-hua Wang (pp. 1097-1103).

The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. The pivotal assets of the antimicrobial peptide include potential for rapid bactericidal activity and low propensity for resistance. The four new antimicrobial hybrid peptides were designed based on peptides LFB15(W4,10), HP(2-20), and cecropin A according to the structure–activity relationship of the amphipathic and cationic antimicrobial peptides. Their structural parameters were accessed by bioinformatics tools, and then two hybrids with the most potential candidates were synthesized. The hybrid peptide LH28 caused an increase in antibiotic activity (MIC₅₀ = 1.56–3.13 μM) against given bacterial strains and did not cause obvious hemolysis of rabbit erythrocytes at concentration of 3.13 μM with effective antimicrobial activity. The results demonstrate that evaluating the structural parameters could be useful for designing novel antimicrobial peptides.

Keywords: Antimicrobial peptides; Design; Structure parameters; Computer-aided method

Isolation of Aspergillus oryzae mutants for heterologous protein production from a double proteinase gene disruptant by Takashi Nemoto; Taisuke Watanabe; Yutaka Mizogami; Jun-ichi Maruyama; Katsuhiko Kitamoto (pp. 1105-1114).

Aspergillus oryzae has attracted much attention as a host for heterologous protein production because of its high secretion ability and safety. However, there have been only a few reports on construction of this organism to improve its properties as a production host. We previously reported that the double disruptant of the proteinase gene (tppA, pepE) improved human lysozyme (HLY) production. In this double disruptant, however, the HLY expression plasmid cannot be removed due to its random integration into the genome. In this study, we re-constructed the tppA pepE disruptant as a host for heterologous protein production. By the use of the tppA pepE disruptant, bovine chymosin (CHY) production was enhanced by 1.9-fold. Moreover, we generated HLY-producing strain from the tppA pepE disruptant by curable niaD marker, and then isolated HLY-hyperproducing mutants using the halo assay based on HLY activity. Subsequently, the niaD-based plasmid for HLY production was cured from the mutants by positive selection. The cured strains (named AUT strains) showed production levels of HLY and CHY that were 2.6- and 3.2-fold higher than those of the wild-type strain, respectively. Thus, the AUT strains are expected to be good hosts for obtaining higher production levels of various heterologous proteins.

Keywords: Aspergillus oryzae ; Heterologous protein production; Protease; Mutant; Lysozyme; Chymosin

Multiple control of the acetate pathway in Lactococcus lactis under aeration by catabolite repression and metabolites by Felix Lopez de Felipe; Philippe Gaudu (pp. 1115-1122).

To explore the factors controlling metabolite formation under aeration in Lactococcus lactis, metabolic patterns, enzymatic activities, and transcriptional profiles of genes involved in the aerobic pathway for acetate anabolism were compared between a parental L. lactis strain and its NADH-oxidase-overproducer derivative. Deregulated catabolite repression mutans in the ccpA or pstH genes, encoding CcpA or its co-activator HPr, respectively, were compared with a parental strain, as well. Although the NADH-oxidase activity was derepressed in ccpA, but not in the pstH background, a mixed fermentation was displayed by either mutant, with a higher acetate production in the pstH variant. Moreover, transcription of genes encoding phosphotransacetylase and acetate kinase were derepressed, and the corresponding enzymatic activities increased, in both catabolite repression mutants. These results and the dependence on carbon source for acetate production in the NADH-oxidase-overproducer support the conclusion that catabolite repression, rather than NADH oxidation, plays a critical role to control acetate production. Furthermore, fructose 1,6-bisphosphate influenced the in vitro phosphotransacetylase and acetate kinase activities, while the former was sensitive to diacetyl. Our study strongly supports the model that, under aerobic conditions, the homolactic fermentation in L. lactis MG1363 is maintained by CcpA-mediated repression of mixed acid fermentation.

Keywords: Lactococcus lactis ; Aerobic conditions; Catabolite repression; CcpA; Regulation; NADH-oxidase

Transcriptional profiles of response to terbinafine in Trichophyton rubrum by Wenliang Zhang; Lu Yu; Jian Yang; Lingling Wang; Junping Peng; Qi Jin (pp. 1123-1130).

Trichophyton rubrum is a good model for the study of human pathogenic filamentous fungi. The antifungal agent terbinafine (TRB) shows specific activity against T. rubrum. To identify the transcriptional profiles of response to TRB in T. rubrum, a cDNA microarray was constructed from the expressed sequence tags of different phase cDNA libraries and transcriptional profiles of the response to TRB were determined. Exposure to TRB decreased the transcription of some genes involved in the ergosterol biosynthesis pathway, including ERG2, ERG4, ERG24, and ERG25, and induced the expression of some genes involved in lipid metabolism such as ERG10, ERG13, and INO1. TRB affected transcription of multidrug-resistance genes and some genes encoding ribosomal proteins.

Keywords: Transcriptional profiles; Trichophyton rubrum ; Terbinafine; Microarray; Antifungal

A putative azoreductase gene is involved in the Shewanella oneidensis response to heavy metal stress by Irina Mugerfeld; Brittany A. Law; Gene S. Wickham; Dorothea K. Thompson (pp. 1131-1141).

The Shewanella oneidensis MR-1 gene SO3585, which is annotated as a putative flavin mononucleotide-dependent azoreductase, shares 28% sequence identity with Bacillus subtilis azoreductase and Pseudomonas putida ChrR, a soluble flavoprotein exhibiting chromate reductase activity. Reverse transcription polymerase chain reaction demonstrated that the SO3585 gene is co-transcribed with two downstream open reading frames: SO3586 (a glyoxalase family protein) and SO3587 (a predicted membrane-associated hypothetical protein). The transcriptional start site of the so3585 transcript was localized using 5′ rapid amplification of complementary DNA ends analysis. To investigate the cellular function of SO3585, an in-frame deletion of the so3585 locus was generated in MR-1, and the phenotype of the resulting mutant was characterized. The so3585 deletion mutant was comparable to the parental strain in its ability to decolorize two sulfonated azo dyes (Orange II, Direct Blue 15) under aerobic conditions. By contrast, growth of the so3585 deletion mutant was sensitive to different exogenous transition heavy metals [Cr(VI), Cd(II), Cu(II), and Zn(II)], while the most severe growth deficiencies were observed in the presence of Cd(II) and Cu(II). In addition, the rate of extracellular chromate disappearance by the deletion strain was initially impaired, although both the so3585 mutant and MR-1 wild type reduced Cr(VI) within the same time period.

Keywords: Shewanella ; Putative azoreductase; Metal stress

The role of oxygen in yeast metabolism during high cell density brewery fermentations by P. J. Verbelen; S. M. G. Saerens; S. E. Van Mulders; F. Delvaux; F. R. Delvaux (pp. 1143-1156).

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e., higher inoculum size). However, the decreased yeast net growth observed in these high cell density fermentations can have a negative impact on the physiological stability throughout subsequent yeast generations. The use of different oxygen conditions (wort aeration, wort oxygenation, yeast preoxygenation) was investigated to improve the growth yield during high cell density fermentations and yeast metabolic and physiological parameters were assessed systematically. Together with a higher extent of growth (dependent on the applied oxygen conditions), the fermentation power and the formation of unsaturated fatty acids were also affected. Wort oxygenation had a significant decreasing effect on the formation of esters, which was caused by a decreased expression of the alcohol acetyl transferase gene ATF1, compared with the other conditions. Lower glycogen and trehalose levels at the end of fermentation were observed in case of the high cell density fermentations with oxygenated wort and the reference fermentation. The expression levels of BAP2 (encoding the branched chain amino acid permease), ERG1 (encoding squalene epoxidase), and the stress responsive gene HSP12 were predominantly influenced by the high cell concentrations, while OLE1 (encoding the fatty acid desaturase) and the oxidative stress responsive genes SOD1 and CTT1 were mainly affected by the oxygen availability per cell. These results demonstrate that optimisation of high cell density fermentations could be achieved by improving the oxygen conditions, without drastically affecting the physiological condition of the yeast and beer quality.

Keywords: Fermentation; Brewer’s yeast; Yeast physiology; Stress response; Oxygen; Flavour compounds

Efficient constitutive expression of chitinase in the mother cell of Bacillus thuringiensis and its potential to enhance the toxicity of Cry1Ac protoxin by S. B. Hu; P. Liu; X. Z. Ding; L. Yan; Y. J. Sun; Y. M. Zhang; W. P. Li; L. Q. Xia (pp. 1157-1167).

Previous studies revealed that chitinase could enhance the insecticidal activity of Bacillus thuringiensis and it has been used in combination with B. thuringiensis widely. However, the expression of B. thuringiensis chitinase is rather low and needs induction by chitin, which limits its field application. It would make sense to constitutively express the chitinase at a sufficiently high level to offer advantages in biological control of pests. In this study, a signal peptide-encoding sequence-deleted chitinase gene from B. thuringiensis strain 4.0718 under the control of dual overlapping promoters plus Shine–Dalgarno sequence and terminator sequence of cry1Ac3 gene was cloned into shuttle vector pHT315 and introduced into an acrystalliferous B. thuringiensis strain Cry⁻B. The recombinant plasmid was stably maintained over 240 generations in Cry⁻B. Chitinase was overexpressed within the sporangial mother cells in the form of spherical crystal-like inclusion bodies. The chitinase inclusions could be solubilized and exhibit chitinolytic activity in 30 mmol l⁻¹ Na₂CO₃–0.2% β-mercaptoethanol buffer at a wide range of alkaline pH values, and what’s more, the chitinase inclusions potentiated the insecticidal effect of Cry1Ac protoxin when used against larvae of Spodoptera exigua and Helicoverpa armigera.

Keywords: Insecticidal crystal proteins; Biological control of pests; Insecticidal activity; Inclusion body; Solubility property; Chitinolytic activity

Characterization and quantification of class 1 integrons and associated gene cassettes in sewage treatment plants by Xu-Xiang Zhang; Tong Zhang; Ming Zhang; Herbert H. P. Fang; Shu-Pei Cheng (pp. 1169-1177).

Class 1 integrons and gene cassettes containing antibiotic resistance genes (ARGs) in five different sewage treatment plants (STPs) were characterized and quantified using polymerase chain reaction (PCR), sequencing, and quantitative real-time PCR (qRT-PCR) in this study. Class 1 integronase gene (intI1) was found commonly occurring in all of activated sludge samples from the five STPs, as well as in influent and effluent of two STPs at Hong Kong. One hundred and nine lactose-fermenting Enterobacteriaceae (LFE) strains were isolated from activated sludge of Shatin STP. Among them, 36 strains (33.0%) were found to carry class 1 integrons. PCR assays showed that 11 of the 36 intI1-carrying isolates harbored a common type of gene cassette array of about 1,600 bps, as well as the static genes (sulI and qacEΔ1) on class 1 integrons. This gene cassette array was found phylogenetically close to antibiotic resistance genes dfr17 and aadA5, encoding dihydrofolate reductase conferring resistance to trimethoprim and adenylyltransferase conferring resistance to spectinomycin/streptomycin, respectively. Antimicrobial susceptibility analysis demonstrated that all the 11 LFEs carrying gene cassette were multi-resistant, especially having common resistance to trimethoprim and streptomycin. qRT-PCR assay showed that genes copies of both class 1 integron and the gene cassette varied significantly among the activated sludge sampled from different STPs, at different time points or different treatment steps. More than 90% of class 1 integrons and the gene cassette were removed by activated sludge processes in two STPs, while the disinfection process removed 94% integron and 77% gene cassette in one STP.

Keywords: Antibiotic resistance gene; Gene cassette; Integron; qRT-PCR; Sewage treatment plant

Construction of recombinant Nitrosomonas europaea expressing green fluorescent protein in response to co-oxidation of chloroform by Barbara O. Gvakharia; Peter J. Bottomley; Daniel J. Arp; Luis A. Sayavedra-Soto (pp. 1179-1185).

Transcriptional fusions with gfp driven by the promoter region of mbla (NE2571) in pPRO/mbla4 and clpB (NE2402) in pPRO/clpb7 were used to transform the ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718). The two genes were chosen because their transcript levels were found at much higher levels in N. europaea in response to oxidation of chloroform and chloromethane. In N. europaea transformed with pPRO/mbla4, green fluorescent protein (GFP)-dependent fluorescence increased from 3- to 18-fold above control levels in response to increasing chloroform concentrations (7 to 28 μM), and from 8- to 10-fold in response to increasing hydrogen peroxide concentrations (2.5–7.5 mM). The GFP-dependent fluorescence of N. europaea transformed with pPRO/clpb7 also showed an increase of 6- to 10-fold in response to chloroform (28–100 μM) but did not respond to H₂O₂. Our data provide proof of concept that biosensors can be fabricated in ammonia-oxidizing bacteria using “sentinel” genes that up-regulate in response to stress caused either by co-oxidation of chlorinated solvents or by the presence of H₂O₂. The fabricated biosensors had a consistent concentration-dependent response to chloroform; however, these did not respond to other chlorinated compounds that cause similar cellular stress.

Keywords: Nitrosomonas europaea ; Biosensor; Green fluorescent protein; Chloroform; Hydrogen peroxide

Application of random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis for high-resolution identification of Monascus strains by Naoya Shinzato; Tomoyuki Namihira; Yasutomo Tamaki; Masatoshi Tsukahara; Toru Matsui (pp. 1187-1193).

Monascus fungi are commonly used for a variety of food products in Asia, and are also known to produce some biologically active compounds. Since the use of Monascus is expected to increase in food industries, strain-level identification and management of Monascus will be needed in the near future. In the present study, random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis was applied for this purpose. Evaluations of the analysis stability revealed that reproducible results could be obtained, although template DNA fragmentation could influence the resulting RAPD pattern. RAPD analysis using 15 Monascus strains consisting of four species, M. ruber, M. pilosus, M. purpureus, and M. kaoliang showed that each strain generated a unique RAPD pattern, which allows strain-level identification of Monascus. In addition, the phylogenetic tree constructed from RAPD patterns reflected M. ruber–M. pilosus and M. purpureus–M. kaoliang clusters inferred from both ITS and β-tubulin gene sequences, which indicated that the RAPD pattern could reflect their phylogenetic traits to a certain extent. On the other hand, RAPD analysis did not support the monophyletic clustering of the four Monascus species used in this study, which suggests the necessity of reexamination of species boundaries in Monascus.

Keywords: Monascus ; Random amplified polymorphic DNA (RAPD); Microchip electrophoresis; Internal transcribed spacer; β-tubulin

A T7 RNA polymerase-dependent gene expression system for Bacillus megaterium by Martin Gamer; David Fröde; Rebekka Biedendieck; Simon Stammen; Dieter Jahn (pp. 1195-1203).

Gene expression systems based on the RNA polymerase of the bacteriophage T7 are often the ultimate choice for the high level production of recombinant proteins. During the last decade, the Gram-positive bacterium Bacillus megaterium was established as a useful host for the intra- and extracellular production of heterologous proteins. In this paper, we report on the development of a T7 RNA polymerase-dependent expression system for B. megaterium. The system was evaluated for cytosolic and secretory protein production with green fluorescent protein (GFP) from Aequoria victoria as intracellular and Lactobacillus reuteri levansucrase as extracellular model protein. GFP accumulated rapidly at high levels up to 50 mg/l shake flask culture intracellularly after induction of T7 RNA polymerase gene expression. The addition of rifampicin for the inhibition of B. megaterium RNA polymerase led to an increased stability of GFP. L. reuteri levansucrase was also successfully produced and secreted (up to 20 U/l) into the culture supernatant. However, parallel intracellular accumulation of the protein indicated limitations affiliated with the Sec-dependent protein translocation process.

Keywords: Bacillus megaterium ; T7 RNA polymerase; Recombinant protein production

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Applied Microbiology and Biotechnology (v.82, #6)

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Applied Microbiology and Biotechnology (v.82, #6)