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Applied Microbiology and Biotechnology (v.84, #4)
Biotechnological production of itaconic acid and its biosynthesis in Aspergillus terreus by Mitsuyasu Okabe; Dwiarti Lies; Shin Kanamasa; Enoch Y. Park (pp. 597-606).
More than 80,000 tons of itaconic acid (IA) is produced worldwide each year and is sold at a price of around US$ 2/kg. The IA production yield from sugar is higher than 80 g/l. The widespread use of IA in synthetic resins, synthetic fibers, plastics, rubbers, surfactants, and oil additives has resulted in an increased demand for this product. However, at present, the IA production capacity exceeds the demand because this product has a restricted range of applications. Studies have been actively conducted in different biomedical fields—dental, ophthalmic, and drug delivery—to extend the range of applications of IA. Recently, many researchers have attempted to replace the carbon source used for microbial production of IA with cheaper alternative substrates. However, there is still a need for new biotechnology innovations that would help to reduce the production costs, such as innovative process development and strain improvement to allow the use of a low-quality carbon source. In this short review, we discuss the following aspects of IA production: strain improvement, process development, identification of the key enzyme cis-aconitic acid decarboxylase (CAD) in the IA metabolic pathway, metabolic importance of CAD, and new applications of IA.
Keywords: Itaconic acid; Aspergillus terreus ; cis-Aconitic decarboxylase; Biorefinery
Biotechnological production of itaconic acid and its biosynthesis in Aspergillus terreus by Mitsuyasu Okabe; Dwiarti Lies; Shin Kanamasa; Enoch Y. Park (pp. 597-606).
More than 80,000 tons of itaconic acid (IA) is produced worldwide each year and is sold at a price of around US$ 2/kg. The IA production yield from sugar is higher than 80 g/l. The widespread use of IA in synthetic resins, synthetic fibers, plastics, rubbers, surfactants, and oil additives has resulted in an increased demand for this product. However, at present, the IA production capacity exceeds the demand because this product has a restricted range of applications. Studies have been actively conducted in different biomedical fields—dental, ophthalmic, and drug delivery—to extend the range of applications of IA. Recently, many researchers have attempted to replace the carbon source used for microbial production of IA with cheaper alternative substrates. However, there is still a need for new biotechnology innovations that would help to reduce the production costs, such as innovative process development and strain improvement to allow the use of a low-quality carbon source. In this short review, we discuss the following aspects of IA production: strain improvement, process development, identification of the key enzyme cis-aconitic acid decarboxylase (CAD) in the IA metabolic pathway, metabolic importance of CAD, and new applications of IA.
Keywords: Itaconic acid; Aspergillus terreus ; cis-Aconitic decarboxylase; Biorefinery
Analytical studies of silica biomineralization: towards an understanding of silica processing by diatoms by Eike Brunner; Christian Gröger; Katharina Lutz; Patrick Richthammer; Katrin Spinde; Manfred Sumper (pp. 607-616).
Diatoms have continued to attract research interest over a long time. One important reason for this research interest is the amazingly beautiful microstructured and nanostructured patterning of the silica-based diatom cell walls. These materials become increasingly important from the materials science point of view. However, many aspects of diatom cell wall formation and patterning are still not fully understood. The present minireview article summarizes our recent knowledge especially with respect to two major topics related to diatom cell wall formation and patterning: (1) uptake and metabolism of silicon by living diatom cells and (2) understanding of the genetic control of cell wall formation. Analytical techniques as well as recent results concerning these two topics are highlighted in this review.
Keywords: Biomineralization; Diatoms; Microstructure and nanostucture; Silicon metabolism; Analytical methods
Analytical studies of silica biomineralization: towards an understanding of silica processing by diatoms by Eike Brunner; Christian Gröger; Katharina Lutz; Patrick Richthammer; Katrin Spinde; Manfred Sumper (pp. 607-616).
Diatoms have continued to attract research interest over a long time. One important reason for this research interest is the amazingly beautiful microstructured and nanostructured patterning of the silica-based diatom cell walls. These materials become increasingly important from the materials science point of view. However, many aspects of diatom cell wall formation and patterning are still not fully understood. The present minireview article summarizes our recent knowledge especially with respect to two major topics related to diatom cell wall formation and patterning: (1) uptake and metabolism of silicon by living diatom cells and (2) understanding of the genetic control of cell wall formation. Analytical techniques as well as recent results concerning these two topics are highlighted in this review.
Keywords: Biomineralization; Diatoms; Microstructure and nanostucture; Silicon metabolism; Analytical methods
Metabolism of fluoroorganic compounds in microorganisms: impacts for the environment and the production of fine chemicals by Cormac D. Murphy; Benjamin R. Clark; Jessica Amadio (pp. 617-629).
Incorporation of fluorine into an organic compound can favourably alter its physicochemical properties with respect to biological activity, stability and lipophilicity. Accordingly, this element is found in many pharmaceutical and industrial chemicals. Organofluorine compounds are accepted as substrates by many enzymes, and the interactions of microorganisms with these compounds are of relevance to the environment and the fine chemicals industry. On the one hand, the microbial transformation of organofluorines can lead to the generation of toxic compounds that are of environmental concern, yet similar biotransformations can yield difficult-to-synthesise products and intermediates, in particular derivatives of biologically active secondary metabolites. In this paper, we review the historical and recent developments of organofluorine biotransformation in microorganisms and highlight the possibility of using microbes as models of fluorinated drug metabolism in mammals.
Keywords: Biodegradation; F-19 NMR; Biotransformation; Natural products; Drug metabolism; Mutasynthesis
Metabolism of fluoroorganic compounds in microorganisms: impacts for the environment and the production of fine chemicals by Cormac D. Murphy; Benjamin R. Clark; Jessica Amadio (pp. 617-629).
Incorporation of fluorine into an organic compound can favourably alter its physicochemical properties with respect to biological activity, stability and lipophilicity. Accordingly, this element is found in many pharmaceutical and industrial chemicals. Organofluorine compounds are accepted as substrates by many enzymes, and the interactions of microorganisms with these compounds are of relevance to the environment and the fine chemicals industry. On the one hand, the microbial transformation of organofluorines can lead to the generation of toxic compounds that are of environmental concern, yet similar biotransformations can yield difficult-to-synthesise products and intermediates, in particular derivatives of biologically active secondary metabolites. In this paper, we review the historical and recent developments of organofluorine biotransformation in microorganisms and highlight the possibility of using microbes as models of fluorinated drug metabolism in mammals.
Keywords: Biodegradation; F-19 NMR; Biotransformation; Natural products; Drug metabolism; Mutasynthesis
Applications of dimethylallyltryptophan synthases and other indole prenyltransferases for structural modification of natural products by Shu-Ming Li (pp. 631-639).
A series of putative indole prenyltransferase genes could be identified in the genome sequences of different fungal strains including Aspergillus fumigatus and Neosartorya fischeri. The gene products show significant sequence similarities to dimethylallyltryptophan synthases from various fungi. These genes belong to different gene clusters and are involved in the biosynthesis of secondary metabolites. Ten of them were cloned and overexpressed in Escherichia coli and Saccharomyces cerevisiae and proven to be soluble proteins. They catalyse different prenyl transfer reactions onto indole moieties of various substrates and do not require divalent metal ions for their prenyl transfer reactions. These enzymes showed broad substrate specificities towards their aromatic substrates. Diverse simple tryptophan derivatives and tryptophan-containing cyclic dipeptides were accepted by several prenyltransferases as substrates and converted to prenylated derivatives. This feature of substrate flexibility was successfully used for regiospecific and stereospecific synthesis of different indole derivatives.
Keywords: Aspergillus ; Neosartorya ; Chemoenzymatic synthesis; Indole alkaloid; Cyclic dipeptide; Diketopiperazine; Prenyltransferase; Dimethylallyltryptophan synthase; Indole derivatives
Applications of dimethylallyltryptophan synthases and other indole prenyltransferases for structural modification of natural products by Shu-Ming Li (pp. 631-639).
A series of putative indole prenyltransferase genes could be identified in the genome sequences of different fungal strains including Aspergillus fumigatus and Neosartorya fischeri. The gene products show significant sequence similarities to dimethylallyltryptophan synthases from various fungi. These genes belong to different gene clusters and are involved in the biosynthesis of secondary metabolites. Ten of them were cloned and overexpressed in Escherichia coli and Saccharomyces cerevisiae and proven to be soluble proteins. They catalyse different prenyl transfer reactions onto indole moieties of various substrates and do not require divalent metal ions for their prenyl transfer reactions. These enzymes showed broad substrate specificities towards their aromatic substrates. Diverse simple tryptophan derivatives and tryptophan-containing cyclic dipeptides were accepted by several prenyltransferases as substrates and converted to prenylated derivatives. This feature of substrate flexibility was successfully used for regiospecific and stereospecific synthesis of different indole derivatives.
Keywords: Aspergillus ; Neosartorya ; Chemoenzymatic synthesis; Indole alkaloid; Cyclic dipeptide; Diketopiperazine; Prenyltransferase; Dimethylallyltryptophan synthase; Indole derivatives
Application of promoter swapping techniques to control expression of chromosomal genes by William R. McCleary (pp. 641-648).
The ability to control the expression of chromosomal genes is important for many applications, including metabolic engineering and the functional analysis of cellular processes. This mini-review presents recent work on the application of techniques that allow researchers to replace a chromosomal promoter with one designed for a specific level of activity, thereby exerting precise transcriptional control while retaining the natural genetic context of a gene or operon. This technique, termed promoter swapping, involves the creation of a PCR product that encodes a removable antibiotic resistance cassette and an engineered promoter. Short homology sequences on the ends of the PCR fragment target it for homologous recombination with the chromosome catalyzed by phage-derived recombination proteins. After the PCR product is introduced by electroporation into an appropriate acceptor strain, antibiotic resistance selects the desired recombination products. The antibiotic resistance cassette is then removed from the strain by site-specific recombination leaving the engineered promoter precisely positioned upstream of a target gene but downstream of a short scar consisting of a single site-specific recombination site.
Keywords: Promoter swapping; Promoter replacement; Metabolic engineering; Recombineering
Application of promoter swapping techniques to control expression of chromosomal genes by William R. McCleary (pp. 641-648).
The ability to control the expression of chromosomal genes is important for many applications, including metabolic engineering and the functional analysis of cellular processes. This mini-review presents recent work on the application of techniques that allow researchers to replace a chromosomal promoter with one designed for a specific level of activity, thereby exerting precise transcriptional control while retaining the natural genetic context of a gene or operon. This technique, termed promoter swapping, involves the creation of a PCR product that encodes a removable antibiotic resistance cassette and an engineered promoter. Short homology sequences on the ends of the PCR fragment target it for homologous recombination with the chromosome catalyzed by phage-derived recombination proteins. After the PCR product is introduced by electroporation into an appropriate acceptor strain, antibiotic resistance selects the desired recombination products. The antibiotic resistance cassette is then removed from the strain by site-specific recombination leaving the engineered promoter precisely positioned upstream of a target gene but downstream of a short scar consisting of a single site-specific recombination site.
Keywords: Promoter swapping; Promoter replacement; Metabolic engineering; Recombineering
Effect of process parameters on 3-hydroxypropionic acid production from glycerol using a recombinant Escherichia coli by Subramanian Mohan Raj; Chelladurai Rathnasingh; Woo-Chel Jung; Sunghoon Park (pp. 649-657).
The top-valued platform chemical, 3-hydroxypropionic acid (3-HP), has a wide range of industrial applications but its biological production is not well established. Previously, the production of 3-HP from glycerol was demonstrated using a recombinant Escherichia coli strain expressing glycerol dehydratase (dhaB) and aldehyde dehydrogenase (aldH). The present investigation focuses on the effect of the culture conditions on the production of 3-HP from glycerol. The physicochemical parameters, such as pH, IPTG concentration, liquid-to-flask volume ratio, and substrate concentration, were examined in flask-scale experiments and obtained the highest titer of 3-HP at 4.4 g l–1 in 48 h. When a fed-batch process was carried out in a bioreactor under pH-regulated conditions, the recombinant E. coli produced 3-HP at 31 g l–1 in 72 h with a yield of 0.35 mol mol–1 glycerol. The maximum specific rate of 3-HP production was estimated to be 3.41 mmol g–1 cdw h–1 between 12 and 24 h. Other than 3-HP, propionic acid (3.4 g l–1), 1,3-propanediol (2.4 g l–1), and lactic acid (1.6 g l–1) were produced as the major by-products. This paper reports for the first time a commercially meaningful high titer of 3-HP production.
Keywords: 3-Hydroxypropionic acid production; Glycerol; 3-Hydroxypropionaldehyde; Glycerol dehydratase; Aldehyde dehydrogenase; Recombinant E. coli
Effect of process parameters on 3-hydroxypropionic acid production from glycerol using a recombinant Escherichia coli by Subramanian Mohan Raj; Chelladurai Rathnasingh; Woo-Chel Jung; Sunghoon Park (pp. 649-657).
The top-valued platform chemical, 3-hydroxypropionic acid (3-HP), has a wide range of industrial applications but its biological production is not well established. Previously, the production of 3-HP from glycerol was demonstrated using a recombinant Escherichia coli strain expressing glycerol dehydratase (dhaB) and aldehyde dehydrogenase (aldH). The present investigation focuses on the effect of the culture conditions on the production of 3-HP from glycerol. The physicochemical parameters, such as pH, IPTG concentration, liquid-to-flask volume ratio, and substrate concentration, were examined in flask-scale experiments and obtained the highest titer of 3-HP at 4.4 g l–1 in 48 h. When a fed-batch process was carried out in a bioreactor under pH-regulated conditions, the recombinant E. coli produced 3-HP at 31 g l–1 in 72 h with a yield of 0.35 mol mol–1 glycerol. The maximum specific rate of 3-HP production was estimated to be 3.41 mmol g–1 cdw h–1 between 12 and 24 h. Other than 3-HP, propionic acid (3.4 g l–1), 1,3-propanediol (2.4 g l–1), and lactic acid (1.6 g l–1) were produced as the major by-products. This paper reports for the first time a commercially meaningful high titer of 3-HP production.
Keywords: 3-Hydroxypropionic acid production; Glycerol; 3-Hydroxypropionaldehyde; Glycerol dehydratase; Aldehyde dehydrogenase; Recombinant E. coli
High production of 2,3-butanediol from glycerol by Klebsiella pneumoniae G31 by Kaloyan Petrov; Penka Petrova (pp. 659-665).
The microbial production of high amounts of 2,3-butanediol (2,3-BD) from glycerol as a sole carbon source by the Bulgarian isolate Klebsiella pneumoniae G31 was studied in a series of fed-batch processes. The following conditions were evaluated as optimal: micro-aerobic cultivation in modified media, without pH control. Beginning at pH 8, 49.2 g/l of 2,3-BD was produced as negligible concentrations of by-products were received. The pH is the most important factor ruling the 2,3-BD production. Spontaneous pH changes and products formation in time were investigated, performing fermentations with non-controlled pH starting at different initial pH. In lack of external maintenance, the microorganism attempted to control the pH using acetate/2,3-BD alternations of the oxidative pathway of glycerol catabolism, which resulted in pH fluctuations. Thus, the culture secreted 2,3-BD at unequal portions, either allowing or detaining the acetate synthesis. More alkaline initial pH led to enhanced 2,3-BD accumulation as a response to the increased amplitudes of the pH variations. When the pH was maintained constant, the yield of 2,3-BD was very poor. These cultures remained viable only 72 h; whereas, the pH self-controlling cells lived and produced 2,3-BD up to 280 h. In conclusion, the formation of 2,3-BD is a result of an adaptive mechanism of pH self-control, responding to spontaneous pH drops during glycerol fermentation.
Keywords: 2,3-Butanediol; Glycerol; Klebsiella pneumoniae ; pH
High production of 2,3-butanediol from glycerol by Klebsiella pneumoniae G31 by Kaloyan Petrov; Penka Petrova (pp. 659-665).
The microbial production of high amounts of 2,3-butanediol (2,3-BD) from glycerol as a sole carbon source by the Bulgarian isolate Klebsiella pneumoniae G31 was studied in a series of fed-batch processes. The following conditions were evaluated as optimal: micro-aerobic cultivation in modified media, without pH control. Beginning at pH 8, 49.2 g/l of 2,3-BD was produced as negligible concentrations of by-products were received. The pH is the most important factor ruling the 2,3-BD production. Spontaneous pH changes and products formation in time were investigated, performing fermentations with non-controlled pH starting at different initial pH. In lack of external maintenance, the microorganism attempted to control the pH using acetate/2,3-BD alternations of the oxidative pathway of glycerol catabolism, which resulted in pH fluctuations. Thus, the culture secreted 2,3-BD at unequal portions, either allowing or detaining the acetate synthesis. More alkaline initial pH led to enhanced 2,3-BD accumulation as a response to the increased amplitudes of the pH variations. When the pH was maintained constant, the yield of 2,3-BD was very poor. These cultures remained viable only 72 h; whereas, the pH self-controlling cells lived and produced 2,3-BD up to 280 h. In conclusion, the formation of 2,3-BD is a result of an adaptive mechanism of pH self-control, responding to spontaneous pH drops during glycerol fermentation.
Keywords: 2,3-Butanediol; Glycerol; Klebsiella pneumoniae ; pH
Optimization of enzymatic hydrolysis and ethanol fermentation from AFEX-treated rice straw by Cheng Zhong; Ming W. Lau; Venkatesh Balan; Bruce E. Dale; Ying-Jin Yuan (pp. 667-676).
An abundant agricultural residue, rice straw (RS) was pretreated using ammonia fiber expansion (AFEX) process with less than 3% sugar loss. Along with commercial cellulase (Spezyme® CP) at 15 filter paper unit/g of glucan, the addition of Multifect® Xylanase at 2.67 mg protein/g glucan and Multifect® Pectinase at 3.65 mg protein/g glucan was optimized to greatly increase sugar conversion of AFEX-treated RS. During enzymatic hydrolysis even at 6% glucan loading (equivalent to 17.8% solid loading), about 80.6% of glucan and 89.6% of xylan conversions (including monomeric and oligomeric sugars) were achieved. However, oligomeric glucose and xylose accounted for 12.3% of the total glucose and 37.0% of the total xylose, respectively. Comparison among the three ethanologenic strains revealed Saccharomyces cerevisiae 424A(LNH-ST) to be a promising candidate for RS hydrolysate with maximum ethanol metabolic yield of 95.3% and ethanol volumetric productivity of 0.26 g/L/h. The final concentration of ethanol at 37.0 g/L was obtained by S. cerevisiae 424A(LNH-ST) even with low cell density inoculum. A biorefinery combining AFEX pretreatment with S. cerevisiae 424A(LNH-ST) in separate hydrolysis and fermentation could achieve 175.6 g EtOH/kg untreated rice straw at low initial cell density (0.28 g dw/L) without washing pretreated biomass, detoxification, or nutrient supplementation.
Keywords: AFEX pretreatment; Biofuel; Cellulosic ethanol; Mass balance; Saccharification; Biorefinery
Optimization of enzymatic hydrolysis and ethanol fermentation from AFEX-treated rice straw by Cheng Zhong; Ming W. Lau; Venkatesh Balan; Bruce E. Dale; Ying-Jin Yuan (pp. 667-676).
An abundant agricultural residue, rice straw (RS) was pretreated using ammonia fiber expansion (AFEX) process with less than 3% sugar loss. Along with commercial cellulase (Spezyme® CP) at 15 filter paper unit/g of glucan, the addition of Multifect® Xylanase at 2.67 mg protein/g glucan and Multifect® Pectinase at 3.65 mg protein/g glucan was optimized to greatly increase sugar conversion of AFEX-treated RS. During enzymatic hydrolysis even at 6% glucan loading (equivalent to 17.8% solid loading), about 80.6% of glucan and 89.6% of xylan conversions (including monomeric and oligomeric sugars) were achieved. However, oligomeric glucose and xylose accounted for 12.3% of the total glucose and 37.0% of the total xylose, respectively. Comparison among the three ethanologenic strains revealed Saccharomyces cerevisiae 424A(LNH-ST) to be a promising candidate for RS hydrolysate with maximum ethanol metabolic yield of 95.3% and ethanol volumetric productivity of 0.26 g/L/h. The final concentration of ethanol at 37.0 g/L was obtained by S. cerevisiae 424A(LNH-ST) even with low cell density inoculum. A biorefinery combining AFEX pretreatment with S. cerevisiae 424A(LNH-ST) in separate hydrolysis and fermentation could achieve 175.6 g EtOH/kg untreated rice straw at low initial cell density (0.28 g dw/L) without washing pretreated biomass, detoxification, or nutrient supplementation.
Keywords: AFEX pretreatment; Biofuel; Cellulosic ethanol; Mass balance; Saccharification; Biorefinery
Enhanced uridine diphosphate N-acetylglucosamine production using whole-cell catalysis by Hanjie Ying; Xiaochun Chen; Haiping Cao; Jian Xiong; Yuan Hong; Jianxin Bai; Zhenjiang Li (pp. 677-683).
Uridine diphosphate N-acetylglucosamine (UDPAG) can be produced by chemical, enzymatic, chemoenzymatic, and fermentative methods. In this study, we used whole-cell catalysis method to produce UDPAG for the first time by Saccharomyces cerevisiae. In order to increase the ATP utilization efficiency and UDPAG conversion yield, the response surface methodology was applied to optimize the whole-cell catalytic conditions for UDPAG production. Firstly, effects of uridine 5′-monophosphate (5′-UMP), glucosamine, vitamin B1, glycerol, magnesium chloride, potassium chloride, temperature, sodium dihydrogen phosphate, sodium acetate, fructose, and pH on UDPAG production were evaluated by a fractional factorial design. Results showed that UDPAG production was mainly affected by sodium dihydrogen phosphate, temperature, and vitamin B1. Then, the concentrations of sodium dihydrogen phosphate and vitamin B1 and temperature were further investigated with a central composite design and response surface analysis. The cultivation conditions to obtain the optimal UDPAG production were determined: sodium dihydrogen phosphate, 31.2 g/L; temperature, 29°C, and vitamin B1, 0.026 g/L. This optimization strategy led to an enhancement of UDPAG production from 2.51 to 4.25 g/L, yield from 44.6% to 75.6% based on the initial 5′-UMP concentration, and ATP utilization efficiency from 7.43% to 12.6%.
Keywords: ATP utilization efficiency; Response surface methodology; Saccharomyces cerevisiae ; Uridine diphosphate N-acetylglucosamine
Enhanced uridine diphosphate N-acetylglucosamine production using whole-cell catalysis by Hanjie Ying; Xiaochun Chen; Haiping Cao; Jian Xiong; Yuan Hong; Jianxin Bai; Zhenjiang Li (pp. 677-683).
Uridine diphosphate N-acetylglucosamine (UDPAG) can be produced by chemical, enzymatic, chemoenzymatic, and fermentative methods. In this study, we used whole-cell catalysis method to produce UDPAG for the first time by Saccharomyces cerevisiae. In order to increase the ATP utilization efficiency and UDPAG conversion yield, the response surface methodology was applied to optimize the whole-cell catalytic conditions for UDPAG production. Firstly, effects of uridine 5′-monophosphate (5′-UMP), glucosamine, vitamin B1, glycerol, magnesium chloride, potassium chloride, temperature, sodium dihydrogen phosphate, sodium acetate, fructose, and pH on UDPAG production were evaluated by a fractional factorial design. Results showed that UDPAG production was mainly affected by sodium dihydrogen phosphate, temperature, and vitamin B1. Then, the concentrations of sodium dihydrogen phosphate and vitamin B1 and temperature were further investigated with a central composite design and response surface analysis. The cultivation conditions to obtain the optimal UDPAG production were determined: sodium dihydrogen phosphate, 31.2 g/L; temperature, 29°C, and vitamin B1, 0.026 g/L. This optimization strategy led to an enhancement of UDPAG production from 2.51 to 4.25 g/L, yield from 44.6% to 75.6% based on the initial 5′-UMP concentration, and ATP utilization efficiency from 7.43% to 12.6%.
Keywords: ATP utilization efficiency; Response surface methodology; Saccharomyces cerevisiae ; Uridine diphosphate N-acetylglucosamine
Gaseous alkene biotransformation and enantioselective epoxyalkane formation by Nocardioides sp. strain JS614 by Carmen R. Owens; Julie K. Karceski; Timothy E. Mattes (pp. 685-692).
Enantiopure epoxides are valuable intermediates in the synthesis of optically pure biologically active fine chemicals (e.g., pharmaceuticals) that are often difficult to produce by chemical approaches. An attractive alternative is biological synthesis by microorganisms expressing stereoselective enzymes. In this study, we investigated the ability of ethene-grown Nocardioides sp. strain JS614 to produce highly enantio-enriched epoxyalkanes via stereoselective monooxygenase-mediated alkene epoxidation. Ethene-grown JS614 cells transformed propene, 1-butene, and trans-2-butene to their corresponding epoxyalkanes at rates ranging from 27.1 to 44.0 nmol/min mg protein. Chiral gas chromatography analysis revealed that R-1,2-epoxypropane, R-1,2-epoxybutane, and trans-2R,3R-epoxybutane were produced in enantiomeric excess (e.e.) of 98%, 74%, and 82%, respectively. Ethene-grown JS614 cells also preferentially transformed trans-2S,3S-epoxybutane from a racemic mixture, but could not resolve racemic 1,2-epoxypropane. Glucose facilitated increased epoxyalkane production by ethene-grown JS614 cells. However, after 22 h of propene biotransformation with 20 mM glucose, 84% of ethene-grown JS614 cells lost membrane integrity and the remaining live cells were not viable. Propene biotransformation by JS614 was extended beyond 22 h and 54% more epoxypropane was produced when cells were resuspended in fresh buffer + glucose at 8-h intervals. We conclude that JS614 is a promising new biocatalyst for applications that involve enantiopure epoxide production.
Keywords: Biocatalysis; Nocardioides ; Epoxyalkanes; Toxicity; Alkene monooxygenase; Stereoselectivity
Gaseous alkene biotransformation and enantioselective epoxyalkane formation by Nocardioides sp. strain JS614 by Carmen R. Owens; Julie K. Karceski; Timothy E. Mattes (pp. 685-692).
Enantiopure epoxides are valuable intermediates in the synthesis of optically pure biologically active fine chemicals (e.g., pharmaceuticals) that are often difficult to produce by chemical approaches. An attractive alternative is biological synthesis by microorganisms expressing stereoselective enzymes. In this study, we investigated the ability of ethene-grown Nocardioides sp. strain JS614 to produce highly enantio-enriched epoxyalkanes via stereoselective monooxygenase-mediated alkene epoxidation. Ethene-grown JS614 cells transformed propene, 1-butene, and trans-2-butene to their corresponding epoxyalkanes at rates ranging from 27.1 to 44.0 nmol/min mg protein. Chiral gas chromatography analysis revealed that R-1,2-epoxypropane, R-1,2-epoxybutane, and trans-2R,3R-epoxybutane were produced in enantiomeric excess (e.e.) of 98%, 74%, and 82%, respectively. Ethene-grown JS614 cells also preferentially transformed trans-2S,3S-epoxybutane from a racemic mixture, but could not resolve racemic 1,2-epoxypropane. Glucose facilitated increased epoxyalkane production by ethene-grown JS614 cells. However, after 22 h of propene biotransformation with 20 mM glucose, 84% of ethene-grown JS614 cells lost membrane integrity and the remaining live cells were not viable. Propene biotransformation by JS614 was extended beyond 22 h and 54% more epoxypropane was produced when cells were resuspended in fresh buffer + glucose at 8-h intervals. We conclude that JS614 is a promising new biocatalyst for applications that involve enantiopure epoxide production.
Keywords: Biocatalysis; Nocardioides ; Epoxyalkanes; Toxicity; Alkene monooxygenase; Stereoselectivity
CHO-recombinant human growth hormone as a protease sensitive reporter protein by Renate Kunert; Willibald Steinfellner; Friedrich Altmann; Jakob Wallner; Hermann Katinger; Karola Vorauer-Uhl (pp. 693-699).
Protein-free media are gaining more and more interest in mammalian cell culture technology. However, the range of commercially available protein-free media is wide, but lack of serum causes the lack of various substances (Keenan et al. in Cytotechnology, 50(1–3):49–56, 2006) which must be substituted case by case. Details on the composition of protein-free media are often unavailable or inaccessible in some cases, and as a consequence, there is an obvious need for testing procedures in order to evaluate the various commercialised products for their performance. Additionally, negative effects of tryptic meat digests on product quality have been reported in the literature (Gu et al. in Biotech Bioeng 56 (4):353–341, 1997). In the present studies of comparing various protein-free media for their suitability in propagation of recombinant CHO cells expressing human growth hormone (hGH), we have found somatotropin to be an excellent candidate for detection of protease activity. Somatotropin contains protease recognition sites for numerous proteases located around amino-acid residues 134–150. In this study, we demonstrate highly specific cleavage of recombinant hGH during batch cultivation. Analysis of the digested molecule was then performed by convergent methods like SDS-PAGE, HPLC and mass spectroscopy, and the results indicate hGH to be an ideal candidate for media and component screening in mammalian cell culture.
Keywords: Human growth hormone; Somatotropin; Recombinant expression; Two-chain form; Protease; CHO
CHO-recombinant human growth hormone as a protease sensitive reporter protein by Renate Kunert; Willibald Steinfellner; Friedrich Altmann; Jakob Wallner; Hermann Katinger; Karola Vorauer-Uhl (pp. 693-699).
Protein-free media are gaining more and more interest in mammalian cell culture technology. However, the range of commercially available protein-free media is wide, but lack of serum causes the lack of various substances (Keenan et al. in Cytotechnology, 50(1–3):49–56, 2006) which must be substituted case by case. Details on the composition of protein-free media are often unavailable or inaccessible in some cases, and as a consequence, there is an obvious need for testing procedures in order to evaluate the various commercialised products for their performance. Additionally, negative effects of tryptic meat digests on product quality have been reported in the literature (Gu et al. in Biotech Bioeng 56 (4):353–341, 1997). In the present studies of comparing various protein-free media for their suitability in propagation of recombinant CHO cells expressing human growth hormone (hGH), we have found somatotropin to be an excellent candidate for detection of protease activity. Somatotropin contains protease recognition sites for numerous proteases located around amino-acid residues 134–150. In this study, we demonstrate highly specific cleavage of recombinant hGH during batch cultivation. Analysis of the digested molecule was then performed by convergent methods like SDS-PAGE, HPLC and mass spectroscopy, and the results indicate hGH to be an ideal candidate for media and component screening in mammalian cell culture.
Keywords: Human growth hormone; Somatotropin; Recombinant expression; Two-chain form; Protease; CHO
Characterization of a novel cry8 gene specific to Melolonthidae pests: Holotrichia oblita and Holotrichia parallela by Changlong Shu; Guixin Yan; Rongyan Wang; Jie Zhang; Shuliang Feng; Dafang Huang; Fuping Song (pp. 701-707).
A new polymerase chain reaction–restriction fragment length polymorphism method for the identification of cry8-type genes from Bacillus thuringiensis has been established by designing a pair of new universal primers. By this method, a novel gene, cry8Ga1, encoding a polypeptide of 1,157 amino acids with a deduced molecular mass of 131.2 kDa was identified and cloned from B. thuringiensis HBF-18. Recombinant B. thuringiensis strain HD8G, harboring cry8Ga1, has insecticidal activity against larvae of Melolonthidae pests: Holotrichia oblita and Holotrichia parallela. This is the first report of a Cry toxin that has insecticidal activity to Melolonthidae pest H. oblita.
Keywords: PCR–RFLP; Bacillus thuringiensis ; Cry8Ga; Holotrichia oblita ; Holotrichia parallela
Characterization of a novel cry8 gene specific to Melolonthidae pests: Holotrichia oblita and Holotrichia parallela by Changlong Shu; Guixin Yan; Rongyan Wang; Jie Zhang; Shuliang Feng; Dafang Huang; Fuping Song (pp. 701-707).
A new polymerase chain reaction–restriction fragment length polymorphism method for the identification of cry8-type genes from Bacillus thuringiensis has been established by designing a pair of new universal primers. By this method, a novel gene, cry8Ga1, encoding a polypeptide of 1,157 amino acids with a deduced molecular mass of 131.2 kDa was identified and cloned from B. thuringiensis HBF-18. Recombinant B. thuringiensis strain HD8G, harboring cry8Ga1, has insecticidal activity against larvae of Melolonthidae pests: Holotrichia oblita and Holotrichia parallela. This is the first report of a Cry toxin that has insecticidal activity to Melolonthidae pest H. oblita.
Keywords: PCR–RFLP; Bacillus thuringiensis ; Cry8Ga; Holotrichia oblita ; Holotrichia parallela
Identification of a novel fatty acid elongase with a wide substrate specificity from arachidonic acid-producing fungus Mortierella alpina 1S-4 by Eiji Sakuradani; Masutoshi Nojiri; Haruna Suzuki; Sakayu Shimizu (pp. 709-716).
The isolation and characterization of a gene (MALCE1) that encodes a fatty acid elongase from arachidonic acid-producing fungus Mortierella alpina 1S-4 are described. MALCE1 was confirmed to encode a fatty acid elongase by its expression in yeast Saccharomyces cerevisiae, resulting in the accumulation of 18-, 19-, and 20-carbon monounsaturated fatty acids and eicosanoic acid. Furthermore, the MALCE1 yeast transformant efficiently elongated exogenous 9-hexadecenoic acid, 9,12-octadecadienoic acid, and 9,12,15-octadecatrienoic acid. The MALCE1 gene-silenced strain obtained from M. alpina 1S-4 exhibited a low content of octadecanoic acid and a high content of hexadecanoic acid, compared with those in the wild strain. The enzyme encoded by MALCE1 was demonstrated to be involved in the conversion of hexadecanoic acid to octadecanoic acid, its main role in M. alpina 1S-4.
Keywords: Mortierella alpina ; Fatty acid elongase; RNA interference; Gene expression; Polyunsaturated fatty acid
Identification of a novel fatty acid elongase with a wide substrate specificity from arachidonic acid-producing fungus Mortierella alpina 1S-4 by Eiji Sakuradani; Masutoshi Nojiri; Haruna Suzuki; Sakayu Shimizu (pp. 709-716).
The isolation and characterization of a gene (MALCE1) that encodes a fatty acid elongase from arachidonic acid-producing fungus Mortierella alpina 1S-4 are described. MALCE1 was confirmed to encode a fatty acid elongase by its expression in yeast Saccharomyces cerevisiae, resulting in the accumulation of 18-, 19-, and 20-carbon monounsaturated fatty acids and eicosanoic acid. Furthermore, the MALCE1 yeast transformant efficiently elongated exogenous 9-hexadecenoic acid, 9,12-octadecadienoic acid, and 9,12,15-octadecatrienoic acid. The MALCE1 gene-silenced strain obtained from M. alpina 1S-4 exhibited a low content of octadecanoic acid and a high content of hexadecanoic acid, compared with those in the wild strain. The enzyme encoded by MALCE1 was demonstrated to be involved in the conversion of hexadecanoic acid to octadecanoic acid, its main role in M. alpina 1S-4.
Keywords: Mortierella alpina ; Fatty acid elongase; RNA interference; Gene expression; Polyunsaturated fatty acid
Overproduction of poly-β-hydroxybutyrate in the Azotobacter vinelandii mutant that does not express small RNA ArrF by Rajkumar Pyla; Tae-Jo Kim; Juan L. Silva; Yean-Sung Jung (pp. 717-724).
Azotobacter vinelandii contains an iron-regulatory small RNA ArrF whose expression is dependent upon the levels of iron and ferric uptake regulator. The deletion of this ArrF-encoding gene resulted in a 300-fold increase in the production of poly-β-hydroxybutyrate (PHB), a polymer of industrial importance. This ∆arrF mutant exhibited wild-type growth and growth-associated PHB production. Limited iron and aeration elevated the PHB production in the mutant as well as wild type. Real-time RT-PCR revealed that phbB, phbA, and phbC were upregulated ∼61-, 18-, and eightfold, respectively, in the mutant. The phbR transcript of the activator PhbR for this operon was also ∼11 times more abundant. The analysis of phbR transcript predicted a region of complementarity near its Shine–Dalgarno sequence that could potentially basepair with the conserved region of ArrF. These results suggest that ArrF represses the expression of PhbR in an antisense manner and derepression of this activator in the mutant elevates the expression of phbB, phbA, and phbC, resulting in the PHB overproduction.
Keywords: Polyhydroxybutyrate; Azotobacter vinelandii ; Small RNA; ArrF; Iron; Aeration
Overproduction of poly-β-hydroxybutyrate in the Azotobacter vinelandii mutant that does not express small RNA ArrF by Rajkumar Pyla; Tae-Jo Kim; Juan L. Silva; Yean-Sung Jung (pp. 717-724).
Azotobacter vinelandii contains an iron-regulatory small RNA ArrF whose expression is dependent upon the levels of iron and ferric uptake regulator. The deletion of this ArrF-encoding gene resulted in a 300-fold increase in the production of poly-β-hydroxybutyrate (PHB), a polymer of industrial importance. This ∆arrF mutant exhibited wild-type growth and growth-associated PHB production. Limited iron and aeration elevated the PHB production in the mutant as well as wild type. Real-time RT-PCR revealed that phbB, phbA, and phbC were upregulated ∼61-, 18-, and eightfold, respectively, in the mutant. The phbR transcript of the activator PhbR for this operon was also ∼11 times more abundant. The analysis of phbR transcript predicted a region of complementarity near its Shine–Dalgarno sequence that could potentially basepair with the conserved region of ArrF. These results suggest that ArrF represses the expression of PhbR in an antisense manner and derepression of this activator in the mutant elevates the expression of phbB, phbA, and phbC, resulting in the PHB overproduction.
Keywords: Polyhydroxybutyrate; Azotobacter vinelandii ; Small RNA; ArrF; Iron; Aeration
In vitro selection of scFv and its production: an application of mRNA display and wheat embryo cell-free and E. coli cell production system by Tatsuro Shibui; Teruaki Kobayashi; Keiichiro Kanatani; Hirohisa Koga; Satoru Misawa; Tetsu Isomura; Tooru Sasaki (pp. 725-732).
Synthetic DNA libraries encoding human antibody VL and VH fragments were designed, constructed, and enriched using mRNA display. The enriched libraries were then combined to construct a scFv library for mRNA display. Sequencing revealed that 46% of the library coded for full-length scFvs. Considering the number of molecules used in mRNA display, the size of the library displayed was calculated to be >1010. To verify this, we tried to isolate a scFv against human RANK. A scFv was successfully isolated in the sixth round of panning and was synthesized in wheat embryo cell-free (WE) and Escherichia coli cell systems. In the WE system, even though the production level was high, the product was almost soluble. However, in the E. coli system, it was over-produced as inclusion bodies. The inclusion bodies were successfully refolded and showed approximately the same binding affinity as the WE product. These results demonstrate that using mRNA display with synthetic libraries and WE and E. coli cell production systems, a system for in vitro selection and small- to large-scale production of scFvs has been established.
Keywords: mRNA display; Antibody; scFv; Wheat embryo cell-free; E. coli production
In vitro selection of scFv and its production: an application of mRNA display and wheat embryo cell-free and E. coli cell production system by Tatsuro Shibui; Teruaki Kobayashi; Keiichiro Kanatani; Hirohisa Koga; Satoru Misawa; Tetsu Isomura; Tooru Sasaki (pp. 725-732).
Synthetic DNA libraries encoding human antibody VL and VH fragments were designed, constructed, and enriched using mRNA display. The enriched libraries were then combined to construct a scFv library for mRNA display. Sequencing revealed that 46% of the library coded for full-length scFvs. Considering the number of molecules used in mRNA display, the size of the library displayed was calculated to be >1010. To verify this, we tried to isolate a scFv against human RANK. A scFv was successfully isolated in the sixth round of panning and was synthesized in wheat embryo cell-free (WE) and Escherichia coli cell systems. In the WE system, even though the production level was high, the product was almost soluble. However, in the E. coli system, it was over-produced as inclusion bodies. The inclusion bodies were successfully refolded and showed approximately the same binding affinity as the WE product. These results demonstrate that using mRNA display with synthetic libraries and WE and E. coli cell production systems, a system for in vitro selection and small- to large-scale production of scFvs has been established.
Keywords: mRNA display; Antibody; scFv; Wheat embryo cell-free; E. coli production
Development of novel cell surface display in Corynebacterium glutamicum using porin by Toshihiro Tateno; Kazuki Hatada; Tsutomu Tanaka; Hideki Fukuda; Akihiko Kondo (pp. 733-739).
We have developed a novel cell surface display in Corynebacterium glutamicum using porin proteins as anchor proteins. Porins are localized at C. glutamicum mycolic acid layer and exist as a hexamer. We used α-amylase from Streptococcus bovis 148 (AmyA) as a model protein to be displayed on the C. glutamicum cell surface. AmyA was fused to the C terminus of the porins PorB, PorC, or PorH. Expression vectors using fused proteins under the control of the cspB promoter were constructed and introduced into the C. glutamicum Cm strain. Immunostaining microscopy and flow cytometric analysis revealed that PorB-AmyA, PorC-AmyA, and PorH-AmyA were displayed on the C. glutamicum cell surface. AmyA activity was only detected in the cell fraction of C. glutamicum cells that displayed AmyA fused to PorB, PorC or PorH and AmyA activity was not detected in the supernatants of C. glutamicum culture broths after 72 h cultivation. Thus, we have demonstrated that C. glutamicum porins are very efficient anchor proteins for protein display in C. glutamicum.
Keywords: Corynebacterium glutamicum ; Cell surface display; Porin
Development of novel cell surface display in Corynebacterium glutamicum using porin by Toshihiro Tateno; Kazuki Hatada; Tsutomu Tanaka; Hideki Fukuda; Akihiko Kondo (pp. 733-739).
We have developed a novel cell surface display in Corynebacterium glutamicum using porin proteins as anchor proteins. Porins are localized at C. glutamicum mycolic acid layer and exist as a hexamer. We used α-amylase from Streptococcus bovis 148 (AmyA) as a model protein to be displayed on the C. glutamicum cell surface. AmyA was fused to the C terminus of the porins PorB, PorC, or PorH. Expression vectors using fused proteins under the control of the cspB promoter were constructed and introduced into the C. glutamicum Cm strain. Immunostaining microscopy and flow cytometric analysis revealed that PorB-AmyA, PorC-AmyA, and PorH-AmyA were displayed on the C. glutamicum cell surface. AmyA activity was only detected in the cell fraction of C. glutamicum cells that displayed AmyA fused to PorB, PorC or PorH and AmyA activity was not detected in the supernatants of C. glutamicum culture broths after 72 h cultivation. Thus, we have demonstrated that C. glutamicum porins are very efficient anchor proteins for protein display in C. glutamicum.
Keywords: Corynebacterium glutamicum ; Cell surface display; Porin
Lactic acid bacteria as reducing and capping agent for the fast and efficient production of silver nanoparticles by Liesje Sintubin; Wim De Windt; Jan Dick; Jan Mast; David van der Ha; Willy Verstraete; Nico Boon (pp. 741-749).
There is a growing demand for silver-based biocides, including both ionic silver forms and metallic nanosilver. The use of metallic nanosilver, typically chemically produced, faces challenges including particle agglomeration, high costs, and upscaling difficulties . Additionally, there exists a need for the development of a more eco-friendly production of nanosilver. In this study, Gram-positive and Gram-negative bacteria were utilized in the non-enzymatic production of silver nanoparticles via the interaction of silver ions and organic compounds present on the bacterial cell. Only lactic acid bacteria, Lactobacillus spp., Pediococcus pentosaceus, Enterococcus faecium, and Lactococcus garvieae, were able to reduce silver. The nanoparticles of the five best producing Lactobacillus spp. were examined more into detail with transmission electron microscopy. Particle localization inside the cell, the mean particle size, and size distribution were species dependent, with Lactobacillus fermentum having the smallest mean particle size of 11.2 nm, the most narrow size distribution, and most nanoparticles associated with the outside of the cells. Furthermore, influence of pH on the reduction process was investigated. With increasing pH, silver recovery increased as well as the reduction rate as indicated by UV–VIS analyses. This study demonstrated that Lactobacillus spp. can be used for a rapid and efficient production of silver nanoparticles.
Keywords: Metallic silver; Green chemistry; Cell wall; Antimicrobial; Biological synthesis; Probiotic
Lactic acid bacteria as reducing and capping agent for the fast and efficient production of silver nanoparticles by Liesje Sintubin; Wim De Windt; Jan Dick; Jan Mast; David van der Ha; Willy Verstraete; Nico Boon (pp. 741-749).
There is a growing demand for silver-based biocides, including both ionic silver forms and metallic nanosilver. The use of metallic nanosilver, typically chemically produced, faces challenges including particle agglomeration, high costs, and upscaling difficulties . Additionally, there exists a need for the development of a more eco-friendly production of nanosilver. In this study, Gram-positive and Gram-negative bacteria were utilized in the non-enzymatic production of silver nanoparticles via the interaction of silver ions and organic compounds present on the bacterial cell. Only lactic acid bacteria, Lactobacillus spp., Pediococcus pentosaceus, Enterococcus faecium, and Lactococcus garvieae, were able to reduce silver. The nanoparticles of the five best producing Lactobacillus spp. were examined more into detail with transmission electron microscopy. Particle localization inside the cell, the mean particle size, and size distribution were species dependent, with Lactobacillus fermentum having the smallest mean particle size of 11.2 nm, the most narrow size distribution, and most nanoparticles associated with the outside of the cells. Furthermore, influence of pH on the reduction process was investigated. With increasing pH, silver recovery increased as well as the reduction rate as indicated by UV–VIS analyses. This study demonstrated that Lactobacillus spp. can be used for a rapid and efficient production of silver nanoparticles.
Keywords: Metallic silver; Green chemistry; Cell wall; Antimicrobial; Biological synthesis; Probiotic
Carbon fluxes of xylose-consuming Saccharomyces cerevisiae strains are affected differently by NADH and NADPH usage in HMF reduction by João R. M. Almeida; Magnus Bertilsson; Bärbel Hahn-Hägerdal; Gunnar Lidén; Marie-F. Gorwa-Grauslund (pp. 751-761).
Industrial Saccharomyces cerevisiae strains able to utilize xylose have been constructed by overexpression of XYL1 and XYL2 genes encoding the NADPH-preferring xylose reductase (XR) and the NAD+-dependent xylitol dehydrogenase (XDH), respectively, from Pichia stipitis. However, the use of different co-factors by XR and XDH leads to NAD+ deficiency followed by xylitol excretion and reduced product yield. The furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural inhibit yeast metabolism, prolong the lag phase, and reduce the ethanol productivity. Recently, genes encoding furaldehyde reductases were identified and their overexpression was shown to improve S. cerevisiae growth and fermentation rate in HMF containing media and in lignocellulosic hydrolysate. In the current study, we constructed a xylose-consuming S. cerevisiae strain using the XR/XDH pathway from P. stipitis. Then, the genes encoding the NADH- and the NADPH-dependent HMF reductases, ADH1-S110P-Y295C and ADH6, respectively, were individually overexpressed in this background. The performance of these strains, which differed in their co-factor usage for HMF reduction, was evaluated under anaerobic conditions in batch fermentation in absence or in presence of HMF. In anaerobic continuous culture, carbon fluxes were obtained for simultaneous xylose consumption and HMF reduction. Our results show that the co-factor used for HMF reduction primarily influenced formation of products other than ethanol, and that NADH-dependent HMF reduction influenced product formation more than NADPH-dependent HMF reduction. In particular, NADH-dependent HMF reduction contributed to carbon conservation so that biomass was produced at the expense of xylitol and glycerol formation.
Keywords: ADH1 ; ADH6 ; Furaldehyde reduction; HMF; Xylose; Lignocellulosic hydrolysate
Carbon fluxes of xylose-consuming Saccharomyces cerevisiae strains are affected differently by NADH and NADPH usage in HMF reduction by João R. M. Almeida; Magnus Bertilsson; Bärbel Hahn-Hägerdal; Gunnar Lidén; Marie-F. Gorwa-Grauslund (pp. 751-761).
Industrial Saccharomyces cerevisiae strains able to utilize xylose have been constructed by overexpression of XYL1 and XYL2 genes encoding the NADPH-preferring xylose reductase (XR) and the NAD+-dependent xylitol dehydrogenase (XDH), respectively, from Pichia stipitis. However, the use of different co-factors by XR and XDH leads to NAD+ deficiency followed by xylitol excretion and reduced product yield. The furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural inhibit yeast metabolism, prolong the lag phase, and reduce the ethanol productivity. Recently, genes encoding furaldehyde reductases were identified and their overexpression was shown to improve S. cerevisiae growth and fermentation rate in HMF containing media and in lignocellulosic hydrolysate. In the current study, we constructed a xylose-consuming S. cerevisiae strain using the XR/XDH pathway from P. stipitis. Then, the genes encoding the NADH- and the NADPH-dependent HMF reductases, ADH1-S110P-Y295C and ADH6, respectively, were individually overexpressed in this background. The performance of these strains, which differed in their co-factor usage for HMF reduction, was evaluated under anaerobic conditions in batch fermentation in absence or in presence of HMF. In anaerobic continuous culture, carbon fluxes were obtained for simultaneous xylose consumption and HMF reduction. Our results show that the co-factor used for HMF reduction primarily influenced formation of products other than ethanol, and that NADH-dependent HMF reduction influenced product formation more than NADPH-dependent HMF reduction. In particular, NADH-dependent HMF reduction contributed to carbon conservation so that biomass was produced at the expense of xylitol and glycerol formation.
Keywords: ADH1 ; ADH6 ; Furaldehyde reduction; HMF; Xylose; Lignocellulosic hydrolysate
The neuroendocrine hormone norepinephrine increases Pseudomonas aeruginosa PA14 virulence through the las quorum-sensing pathway by Manjunath Hegde; Thomas K. Wood; Arul Jayaraman (pp. 763-776).
It has been proposed that the gastrointestinal tract environment containing high levels of neuroendocrine hormones is important for gut-derived Pseudomonas aeruginosa infections. In this study, we report that the hormone norepinephrine increases P. aeruginosa PA14 growth, virulence factor production, invasion of HCT-8 epithelial cells, and swimming motility in a concentration-dependent manner. Transcriptome analysis of P. aeruginosa exposed to 500 µM, but not 50 µM, norepinephrine for 7 h showed that genes involved in the regulation of the virulence determinants pyocyanin, elastase, and the Pseudomonas quinolone signal (PQS, 2-heptyl-3-hydroxy-4-quinolone) were upregulated. The production of rhamnolipids, which are also important in P. aeruginosa infections, was not significantly altered in suspension cultures upon exposure to 500 µM norepinephrine but decreased on semisolid surfaces. Swarming motility, a phenotype that is directly influenced by rhamnolipids, was also decreased upon 500 µM norepinephrine exposure. The increase in the transcriptional activation of lasR but not that of rhlR and the increase in the levels of PQS suggest that the effects of norepinephrine are mediated primarily through the las quorum-sensing pathway. Together, our data strongly suggest that norepinephrine can play an important role in gut-derived infections by increasing the pathogenicity of P. aeruginosa PA14.
Keywords: Gut-derived sepsis; Interkingdom signaling
The neuroendocrine hormone norepinephrine increases Pseudomonas aeruginosa PA14 virulence through the las quorum-sensing pathway by Manjunath Hegde; Thomas K. Wood; Arul Jayaraman (pp. 763-776).
It has been proposed that the gastrointestinal tract environment containing high levels of neuroendocrine hormones is important for gut-derived Pseudomonas aeruginosa infections. In this study, we report that the hormone norepinephrine increases P. aeruginosa PA14 growth, virulence factor production, invasion of HCT-8 epithelial cells, and swimming motility in a concentration-dependent manner. Transcriptome analysis of P. aeruginosa exposed to 500 µM, but not 50 µM, norepinephrine for 7 h showed that genes involved in the regulation of the virulence determinants pyocyanin, elastase, and the Pseudomonas quinolone signal (PQS, 2-heptyl-3-hydroxy-4-quinolone) were upregulated. The production of rhamnolipids, which are also important in P. aeruginosa infections, was not significantly altered in suspension cultures upon exposure to 500 µM norepinephrine but decreased on semisolid surfaces. Swarming motility, a phenotype that is directly influenced by rhamnolipids, was also decreased upon 500 µM norepinephrine exposure. The increase in the transcriptional activation of lasR but not that of rhlR and the increase in the levels of PQS suggest that the effects of norepinephrine are mediated primarily through the las quorum-sensing pathway. Together, our data strongly suggest that norepinephrine can play an important role in gut-derived infections by increasing the pathogenicity of P. aeruginosa PA14.
Keywords: Gut-derived sepsis; Interkingdom signaling
Molecular identification and dynamics of microbial communities in reactor treating organic household waste by Juliana Cardinali-Rezende; Renan B. Debarry; Luis F. D. B. Colturato; Eduardo V. Carneiro; Edmar Chartone-Souza; Andrea M. A. Nascimento (pp. 777-789).
The prokaryotic diversity associated with organic household waste (OHW), leachate (start-up inoculum), and mesophilic anaerobic digestion processes in the degradation of OHW for 44 and 90 days was investigated using a culture-independent approach. Bacterial and archaeal 16S rRNA and mcrA gene clone libraries were constructed from community DNA preparations. Bacterial clones were affiliated with 13 phyla, of which Firmicutes, Proteobacteria, and Bacteroidetes were represented in all libraries, whereas Actinobacteria, Thermotogae, Lentisphaerae, Acidobacteria, Chloroflexi, Cyanobacteria, Synergistetes, Spirochaetes, Deferribacteres, and Deinococcus-Thermus were exclusively identified in a single library. Within the Archaea domain, the Euryarchaeota phylum was the only one represented. Corresponding sequences were associated with the following orders of hydrogenotrophic methanogens: Methanomicrobiales (Methanoculleus genus) and Methanobacteriales (Methanosphaera and Methanobacterium genera). One archaeal clone was not affiliated with any order and may represent a novel taxon. Diversity indices showed greater diversity of Bacteria when compared to methanogenic Archaea.
Keywords: 16S rRNA gene; mcrA gene; Clone library; Anaerobic reactor; Organic household waste; Leachate
Molecular identification and dynamics of microbial communities in reactor treating organic household waste by Juliana Cardinali-Rezende; Renan B. Debarry; Luis F. D. B. Colturato; Eduardo V. Carneiro; Edmar Chartone-Souza; Andrea M. A. Nascimento (pp. 777-789).
The prokaryotic diversity associated with organic household waste (OHW), leachate (start-up inoculum), and mesophilic anaerobic digestion processes in the degradation of OHW for 44 and 90 days was investigated using a culture-independent approach. Bacterial and archaeal 16S rRNA and mcrA gene clone libraries were constructed from community DNA preparations. Bacterial clones were affiliated with 13 phyla, of which Firmicutes, Proteobacteria, and Bacteroidetes were represented in all libraries, whereas Actinobacteria, Thermotogae, Lentisphaerae, Acidobacteria, Chloroflexi, Cyanobacteria, Synergistetes, Spirochaetes, Deferribacteres, and Deinococcus-Thermus were exclusively identified in a single library. Within the Archaea domain, the Euryarchaeota phylum was the only one represented. Corresponding sequences were associated with the following orders of hydrogenotrophic methanogens: Methanomicrobiales (Methanoculleus genus) and Methanobacteriales (Methanosphaera and Methanobacterium genera). One archaeal clone was not affiliated with any order and may represent a novel taxon. Diversity indices showed greater diversity of Bacteria when compared to methanogenic Archaea.
Keywords: 16S rRNA gene; mcrA gene; Clone library; Anaerobic reactor; Organic household waste; Leachate
The role of anaerobic digestion in controlling the release of tetracycline resistance genes and class 1 integrons from municipal wastewater treatment plants by Sudeshna Ghosh; Sara J. Ramsden; Timothy M. LaPara (pp. 791-796).
In this study, the abilities of two anaerobic digestion processes used for sewage sludge stabilization were compared for their ability to reduce the quantities of three genes that encode resistance to tetracycline (tet(A), tet(O), and tet(X)) and one gene involved with integrons (intI1). A two-stage, thermophilic/mesophilic digestion process always resulted in significant decreases in the quantities of tet(X) and intI1, less frequently in decreases of tet(O), and no net decrease in tet(A). The thermophilic stage was primarily responsible for reducing the quantities of these genes, while the subsequent mesophilic stage sometimes caused a rebound in their quantities. In contrast, a conventional anaerobic digestion process rarely caused a significant decrease in the quantities of any of these genes, with significant increases occurring more frequently. Our results demonstrate that anaerobic thermophilic treatment was more efficient in reducing quantities of genes associated with the spread of antibiotic resistance compared to mesophilic digestion.
Keywords: Anaerobic digestion; Antibiotic resistance; Municipal wastewater treatment; Tetracycline resistance
The role of anaerobic digestion in controlling the release of tetracycline resistance genes and class 1 integrons from municipal wastewater treatment plants by Sudeshna Ghosh; Sara J. Ramsden; Timothy M. LaPara (pp. 791-796).
In this study, the abilities of two anaerobic digestion processes used for sewage sludge stabilization were compared for their ability to reduce the quantities of three genes that encode resistance to tetracycline (tet(A), tet(O), and tet(X)) and one gene involved with integrons (intI1). A two-stage, thermophilic/mesophilic digestion process always resulted in significant decreases in the quantities of tet(X) and intI1, less frequently in decreases of tet(O), and no net decrease in tet(A). The thermophilic stage was primarily responsible for reducing the quantities of these genes, while the subsequent mesophilic stage sometimes caused a rebound in their quantities. In contrast, a conventional anaerobic digestion process rarely caused a significant decrease in the quantities of any of these genes, with significant increases occurring more frequently. Our results demonstrate that anaerobic thermophilic treatment was more efficient in reducing quantities of genes associated with the spread of antibiotic resistance compared to mesophilic digestion.
Keywords: Anaerobic digestion; Antibiotic resistance; Municipal wastewater treatment; Tetracycline resistance
The use of bioluminescence stimulant on the immobilized strain, P. putida mt-2 KG1206, with toluene analog inducers and environmental samples by Kyungsok Ko; In Chul Kong (pp. 797-802).
This study was conducted to investigate the applicability of the stimulant conditions for the bioluminescence activity of a recombinant strain of Pseudomonas putida, mt-2 KG1206, when immobilized using alginate polymer. The bioluminescence activity of the immobilized strain was generally approximately three to five times lower than the subcultured strain, and the activity was observed to slowly decrease. These facts may have been caused by several factors, such as the low biomass and the time required for diffusion into the entrapped biomass. Although different inducers produced different degrees of stimulation, immobilized bacteria modified with KNO3 consistently produced more bioluminescence than those treated with sodium lactate, regardless of the inducer chemical tested. Cells treated with KNO3 exhibited 2.8 times greater bioluminescence than that of the control activity. This condition also stimulated the bioluminescence activities of the immobilized bacteria exposed to contaminated groundwater samples. Based on these results, the immobilized KG1206 presented in this research can be used as a portable assay for the purpose of preliminary on-site monitoring of specific inducer contaminants, with subsequent off-site instrumental analysis, suggesting the potential of this immobilized cell for preliminary application in a field-ready bioassay.
Keywords: Bioassay; Bioluminescence; Groundwater; Immobilization; Recombinant strain; Toluene analogs
The use of bioluminescence stimulant on the immobilized strain, P. putida mt-2 KG1206, with toluene analog inducers and environmental samples by Kyungsok Ko; In Chul Kong (pp. 797-802).
This study was conducted to investigate the applicability of the stimulant conditions for the bioluminescence activity of a recombinant strain of Pseudomonas putida, mt-2 KG1206, when immobilized using alginate polymer. The bioluminescence activity of the immobilized strain was generally approximately three to five times lower than the subcultured strain, and the activity was observed to slowly decrease. These facts may have been caused by several factors, such as the low biomass and the time required for diffusion into the entrapped biomass. Although different inducers produced different degrees of stimulation, immobilized bacteria modified with KNO3 consistently produced more bioluminescence than those treated with sodium lactate, regardless of the inducer chemical tested. Cells treated with KNO3 exhibited 2.8 times greater bioluminescence than that of the control activity. This condition also stimulated the bioluminescence activities of the immobilized bacteria exposed to contaminated groundwater samples. Based on these results, the immobilized KG1206 presented in this research can be used as a portable assay for the purpose of preliminary on-site monitoring of specific inducer contaminants, with subsequent off-site instrumental analysis, suggesting the potential of this immobilized cell for preliminary application in a field-ready bioassay.
Keywords: Bioassay; Bioluminescence; Groundwater; Immobilization; Recombinant strain; Toluene analogs