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Applied Microbiology and Biotechnology (v.94, #4)
Downstream reactions and engineering in the microbially reconstituted pathway for Taxol by Ming Jiang; Gregory Stephanopoulos; Blaine A. Pfeifer (pp. 841-849).
Taxol (a trademarked product of Bristol-Myers Squibb) is a complex isoprenoid natural product which has displayed potent anticancer activity. Originally isolated from the Pacific yew tree (Taxus brevifolia), Taxol has been mass-produced through processes reliant on plant-derived biosynthesis. Recently, there have been alternative efforts to reconstitute the biosynthetic process through technically convenient microbial hosts, which offer unmatched growth kinetics and engineering potential. Such an approach is made challenging by the need to successfully introduce the significantly foreign enzymatic steps responsible for eventual biosynthesis. Doing so, however, offers the potential to engineer more efficient and economical production processes and the opportunity to design and produce tailored analog compounds with enhanced properties. This mini review will specifically focus on heterologous biosynthesis as it applies to Taxol with an emphasis on the challenges associated with introducing and reconstituting the downstream reaction steps needed for final bioactivity.
Keywords: Taxol; Natural products; Metabolic engineering; Heterologous biosynthesis; S. cerevisiae ; E. coli
Downstream reactions and engineering in the microbially reconstituted pathway for Taxol by Ming Jiang; Gregory Stephanopoulos; Blaine A. Pfeifer (pp. 841-849).
Taxol (a trademarked product of Bristol-Myers Squibb) is a complex isoprenoid natural product which has displayed potent anticancer activity. Originally isolated from the Pacific yew tree (Taxus brevifolia), Taxol has been mass-produced through processes reliant on plant-derived biosynthesis. Recently, there have been alternative efforts to reconstitute the biosynthetic process through technically convenient microbial hosts, which offer unmatched growth kinetics and engineering potential. Such an approach is made challenging by the need to successfully introduce the significantly foreign enzymatic steps responsible for eventual biosynthesis. Doing so, however, offers the potential to engineer more efficient and economical production processes and the opportunity to design and produce tailored analog compounds with enhanced properties. This mini review will specifically focus on heterologous biosynthesis as it applies to Taxol with an emphasis on the challenges associated with introducing and reconstituting the downstream reaction steps needed for final bioactivity.
Keywords: Taxol; Natural products; Metabolic engineering; Heterologous biosynthesis; S. cerevisiae ; E. coli
Key players and team play: anaerobic microbial communities in hydrocarbon-contaminated aquifers by Sabine Kleinsteuber; Kathleen M. Schleinitz; Carsten Vogt (pp. 851-873).
Biodegradation of anthropogenic pollutants in shallow aquifers is an important microbial ecosystem service which is mainly brought about by indigenous anaerobic microorganisms. For the management of contaminated sites, risk assessment and control of natural attenuation, the assessment of in situ biodegradation and the underlying microbial processes is essential. The development of novel molecular methods, “omics” approaches, and high-throughput techniques has revealed new insight into complex microbial communities and their functions in anoxic environmental systems. This review summarizes recent advances in the application of molecular methods to study anaerobic microbial communities in contaminated terrestrial subsurface ecosystems. We focus on current approaches to analyze composition, dynamics, and functional diversity of subsurface communities, to link identity to activity and metabolic function, and to identify the ecophysiological role of not yet cultured microbes and syntrophic consortia. We discuss recent molecular surveys of contaminated sites from an ecological viewpoint regarding degrader ecotypes, abiotic factors shaping anaerobic communities, and biotic interactions underpinning the importance of microbial cooperation for microbial ecosystem services such as contaminant degradation.
Keywords: Anoxic aquifers; Functional diversity; Stable isotope probing; Single cell analytics; Metagenomics; Syntrophy
Key players and team play: anaerobic microbial communities in hydrocarbon-contaminated aquifers by Sabine Kleinsteuber; Kathleen M. Schleinitz; Carsten Vogt (pp. 851-873).
Biodegradation of anthropogenic pollutants in shallow aquifers is an important microbial ecosystem service which is mainly brought about by indigenous anaerobic microorganisms. For the management of contaminated sites, risk assessment and control of natural attenuation, the assessment of in situ biodegradation and the underlying microbial processes is essential. The development of novel molecular methods, “omics” approaches, and high-throughput techniques has revealed new insight into complex microbial communities and their functions in anoxic environmental systems. This review summarizes recent advances in the application of molecular methods to study anaerobic microbial communities in contaminated terrestrial subsurface ecosystems. We focus on current approaches to analyze composition, dynamics, and functional diversity of subsurface communities, to link identity to activity and metabolic function, and to identify the ecophysiological role of not yet cultured microbes and syntrophic consortia. We discuss recent molecular surveys of contaminated sites from an ecological viewpoint regarding degrader ecotypes, abiotic factors shaping anaerobic communities, and biotic interactions underpinning the importance of microbial cooperation for microbial ecosystem services such as contaminant degradation.
Keywords: Anoxic aquifers; Functional diversity; Stable isotope probing; Single cell analytics; Metagenomics; Syntrophy
Metabolic engineering of Rhizopus oryzae for the production of platform chemicals by Bas J. Meussen; Leo H. de Graaff; Johan P. M. Sanders; Ruud A. Weusthuis (pp. 875-886).
Rhizopus oryzae is a filamentous fungus belonging to the Zygomycetes. It is among others known for its ability to produce the sustainable platform chemicals l-(+)-lactic acid, fumaric acid, and ethanol. During glycolysis, all fermentable carbon sources are metabolized to pyruvate and subsequently distributed over the pathways leading to the formation of these products. These platform chemicals are produced in high yields on a wide range of carbon sources. The yields are in excess of 85 % of the theoretical yield for l-(+)-lactic acid and ethanol and over 65 % for fumaric acid. The study and optimization of the metabolic pathways involved in the production of these compounds requires well-developed metabolic engineering tools and knowledge of the genetic makeup of this organism. This review focuses on the current metabolic engineering techniques available for R. oryzae and their application on the metabolic pathways of the main fermentation products.
Keywords: Rhizopus oryzae ; Metabolic engineering; Metabolic pathways; Transformation; Heterologous gene expression
Metabolic engineering of Rhizopus oryzae for the production of platform chemicals by Bas J. Meussen; Leo H. de Graaff; Johan P. M. Sanders; Ruud A. Weusthuis (pp. 875-886).
Rhizopus oryzae is a filamentous fungus belonging to the Zygomycetes. It is among others known for its ability to produce the sustainable platform chemicals l-(+)-lactic acid, fumaric acid, and ethanol. During glycolysis, all fermentable carbon sources are metabolized to pyruvate and subsequently distributed over the pathways leading to the formation of these products. These platform chemicals are produced in high yields on a wide range of carbon sources. The yields are in excess of 85 % of the theoretical yield for l-(+)-lactic acid and ethanol and over 65 % for fumaric acid. The study and optimization of the metabolic pathways involved in the production of these compounds requires well-developed metabolic engineering tools and knowledge of the genetic makeup of this organism. This review focuses on the current metabolic engineering techniques available for R. oryzae and their application on the metabolic pathways of the main fermentation products.
Keywords: Rhizopus oryzae ; Metabolic engineering; Metabolic pathways; Transformation; Heterologous gene expression
Sialic acid metabolism and sialyltransferases: natural functions and applications by Yanhong Li; Xi Chen (pp. 887-905).
Sialic acids are a family of negatively charged monosaccharides which are commonly presented as the terminal residues in glycans of the glycoconjugates on eukaryotic cell surface or as components of capsular polysaccharides or lipooligosaccharides of some pathogenic bacteria. Due to their important biological and pathological functions, the biosynthesis, activation, transfer, breaking down, and recycle of sialic acids are attracting increasing attention. The understanding of the sialic acid metabolism in eukaryotes and bacteria leads to the development of metabolic engineering approaches for elucidating the important functions of sialic acid in mammalian systems and for large-scale production of sialosides using engineered bacterial cells. As the key enzymes in biosynthesis of sialylated structures, sialyltransferases have been continuously identified from various sources and characterized. Protein crystal structures of seven sialyltransferases have been reported. Wild-type sialyltransferases and their mutants have been applied with or without other sialoside biosynthetic enzymes for producing complex sialic acid-containing oligosaccharides and glycoconjugates. This mini-review focuses on current understanding and applications of sialic acid metabolism and sialyltransferases.
Keywords: Carbohydrate; Metabolism; Sialic acid; Sialoside; Sialyltransferase
Sialic acid metabolism and sialyltransferases: natural functions and applications by Yanhong Li; Xi Chen (pp. 887-905).
Sialic acids are a family of negatively charged monosaccharides which are commonly presented as the terminal residues in glycans of the glycoconjugates on eukaryotic cell surface or as components of capsular polysaccharides or lipooligosaccharides of some pathogenic bacteria. Due to their important biological and pathological functions, the biosynthesis, activation, transfer, breaking down, and recycle of sialic acids are attracting increasing attention. The understanding of the sialic acid metabolism in eukaryotes and bacteria leads to the development of metabolic engineering approaches for elucidating the important functions of sialic acid in mammalian systems and for large-scale production of sialosides using engineered bacterial cells. As the key enzymes in biosynthesis of sialylated structures, sialyltransferases have been continuously identified from various sources and characterized. Protein crystal structures of seven sialyltransferases have been reported. Wild-type sialyltransferases and their mutants have been applied with or without other sialoside biosynthetic enzymes for producing complex sialic acid-containing oligosaccharides and glycoconjugates. This mini-review focuses on current understanding and applications of sialic acid metabolism and sialyltransferases.
Keywords: Carbohydrate; Metabolism; Sialic acid; Sialoside; Sialyltransferase
Antituberculars which target decaprenylphosphoryl-β-D-ribofuranose 2′-oxidase DprE1: state of art by Silvia Buroni; Maria Rosalia Pasca; Ana Luisa de Jesus Lopes Ribeiro; Giulia Degiacomi; Elisabetta Molteni; Giovanna Riccardi (pp. 907-916).
Multidrug resistance is a major barrier in the battle against tuberculosis and still a leading cause of death worldwide. In order to fight this pathogen, two routes are practicable: vaccination or drug treatment. Vaccination against Mycobacterium tuberculosis with the current vaccine Mycobacterium bovis Bacillus Calmette–Guerin is partially successful, being its efficacy variable. A few new tuberculosis vaccines are now in various phases of clinical trials. The emergence of multidrug-resistant strains of M. tuberculosis gave the impulse to discover new effective antitubercular drugs, a few of which are in clinical development. Here we focus on three different classes of very promising antitubercular drugs recently discovered (benzothiazinones, dinitrobenzamides, and benzoquinoxalines) that share the same cellular target: a subunit of the heteromeric decaprenylphosphoryl-β-d-ribose 2′-epimerase, encoded by the dprE1 (or Rv3790) gene. This enzyme is involved in the biosynthesis of d-arabinose which is crucial for the synthesis of the mycobacterial cell wall and essential for the pathogen’s survival.
Keywords: Tuberculosis; Mycobacteria; Vaccines; DprE1; Drug target
RETRACTED ARTICLE: Antituberculars which target decaprenylphosphoryl-β-D-ribofuranose 2′-oxidase DprE1: state of art by Silvia Buroni; Maria Rosalia Pasca; Ana Luisa de Jesus Lopes Ribeiro; Giulia Degiacomi; Elisabetta Molteni; Giovanna Riccardi (pp. 907-916).
Multidrug resistance is a major barrier in the battle against tuberculosis and still a leading cause of death worldwide. In order to fight this pathogen, two routes are practicable: vaccination or drug treatment. Vaccination against Mycobacterium tuberculosis with the current vaccine Mycobacterium bovis Bacillus Calmette–Guerin is partially successful, being its efficacy variable. A few new tuberculosis vaccines are now in various phases of clinical trials. The emergence of multidrug-resistant strains of M. tuberculosis gave the impulse to discover new effective antitubercular drugs, a few of which are in clinical development. Here we focus on three different classes of very promising antitubercular drugs recently discovered (benzothiazinones, dinitrobenzamides, and benzoquinoxalines) that share the same cellular target: a subunit of the heteromeric decaprenylphosphoryl-β-d-ribose 2′-epimerase, encoded by the dprE1 (or Rv3790) gene. This enzyme is involved in the biosynthesis of d-arabinose which is crucial for the synthesis of the mycobacterial cell wall and essential for the pathogen’s survival.
Keywords: Tuberculosis; Mycobacteria; Vaccines; DprE1; Drug target
RETRACTED ARTICLE: Antituberculars which target decaprenylphosphoryl-β-D-ribofuranose 2′-oxidase DprE1: state of art by Silvia Buroni; Maria Rosalia Pasca; Ana Luisa de Jesus Lopes Ribeiro; Giulia Degiacomi; Elisabetta Molteni; Giovanna Riccardi (pp. 907-916).
Multidrug resistance is a major barrier in the battle against tuberculosis and still a leading cause of death worldwide. In order to fight this pathogen, two routes are practicable: vaccination or drug treatment. Vaccination against Mycobacterium tuberculosis with the current vaccine Mycobacterium bovis Bacillus Calmette–Guerin is partially successful, being its efficacy variable. A few new tuberculosis vaccines are now in various phases of clinical trials. The emergence of multidrug-resistant strains of M. tuberculosis gave the impulse to discover new effective antitubercular drugs, a few of which are in clinical development. Here we focus on three different classes of very promising antitubercular drugs recently discovered (benzothiazinones, dinitrobenzamides, and benzoquinoxalines) that share the same cellular target: a subunit of the heteromeric decaprenylphosphoryl-β-d-ribose 2′-epimerase, encoded by the dprE1 (or Rv3790) gene. This enzyme is involved in the biosynthesis of d-arabinose which is crucial for the synthesis of the mycobacterial cell wall and essential for the pathogen’s survival.
Keywords: Tuberculosis; Mycobacteria; Vaccines; DprE1; Drug target
RETRACTED ARTICLE: Antituberculars which target decaprenylphosphoryl-β-D-ribofuranose 2′-oxidase DprE1: state of art by Silvia Buroni; Maria Rosalia Pasca; Ana Luisa de Jesus Lopes Ribeiro; Giulia Degiacomi; Elisabetta Molteni; Giovanna Riccardi (pp. 907-916).
Multidrug resistance is a major barrier in the battle against tuberculosis and still a leading cause of death worldwide. In order to fight this pathogen, two routes are practicable: vaccination or drug treatment. Vaccination against Mycobacterium tuberculosis with the current vaccine Mycobacterium bovis Bacillus Calmette–Guerin is partially successful, being its efficacy variable. A few new tuberculosis vaccines are now in various phases of clinical trials. The emergence of multidrug-resistant strains of M. tuberculosis gave the impulse to discover new effective antitubercular drugs, a few of which are in clinical development. Here we focus on three different classes of very promising antitubercular drugs recently discovered (benzothiazinones, dinitrobenzamides, and benzoquinoxalines) that share the same cellular target: a subunit of the heteromeric decaprenylphosphoryl-β-d-ribose 2′-epimerase, encoded by the dprE1 (or Rv3790) gene. This enzyme is involved in the biosynthesis of d-arabinose which is crucial for the synthesis of the mycobacterial cell wall and essential for the pathogen’s survival.
Keywords: Tuberculosis; Mycobacteria; Vaccines; DprE1; Drug target
Agar degradation by microorganisms and agar-degrading enzymes by Won-Jae Chi; Yong-Keun Chang; Soon-Kwang Hong (pp. 917-930).
Agar is a mixture of heterogeneous galactans, mainly composed of 3,6-anhydro-l-galactoses (or l-galactose-6-sulfates) d-galactoses and l-galactoses (routinely in the forms of 3,6-anhydro-l-galactoses or l-galactose-6-sulfates) alternately linked by β-(1,4) and α-(1,3) linkages. It is a major component of the cell walls of red algae and has been used in a variety of laboratory and industrial applications, owing to its jellifying properties. Many microorganisms that can hydrolyze and metabolize agar as a carbon and energy source have been identified in seawater and marine sediments. Agarolytic microorganisms commonly produce agarases, which catalyze the hydrolysis of agar. Numerous agarases have been identified in microorganisms of various genera. They are classified according to their cleavage pattern into three types—α-agarase, β-agarase, and β-porphyranase. Although, in a broad sense, many other agarases are involved in complete hydrolysis of agar, most of those identified are β-agarases. In this article we review agarolytic microorganisms and their agar-hydrolyzing systems, covering β-agarases as well as α-agarases, α-neoagarobiose hydrolases, and β-porphyranases, with emphasis on the recent discoveries. We also present an overview of the biochemical and structural characteristics of the various types of agarases. Further, we summarize and compare the agar-hydrolyzing systems of two specific microorganisms: Gram-negative Saccharophagus degradans 2–40 and Gram-positive Streptomyces coelicolor A3(2). We conclude with a brief discussion of the importance of agarases and their possible future application in producing oligosaccharides with various nutraceutical activities and in sustainably generating stock chemicals for biorefinement and bioenergy.
Keywords: Agar; Agarose; Porphyran; Agarase; Porphyranase; Agar degradation
Agar degradation by microorganisms and agar-degrading enzymes by Won-Jae Chi; Yong-Keun Chang; Soon-Kwang Hong (pp. 917-930).
Agar is a mixture of heterogeneous galactans, mainly composed of 3,6-anhydro-l-galactoses (or l-galactose-6-sulfates) d-galactoses and l-galactoses (routinely in the forms of 3,6-anhydro-l-galactoses or l-galactose-6-sulfates) alternately linked by β-(1,4) and α-(1,3) linkages. It is a major component of the cell walls of red algae and has been used in a variety of laboratory and industrial applications, owing to its jellifying properties. Many microorganisms that can hydrolyze and metabolize agar as a carbon and energy source have been identified in seawater and marine sediments. Agarolytic microorganisms commonly produce agarases, which catalyze the hydrolysis of agar. Numerous agarases have been identified in microorganisms of various genera. They are classified according to their cleavage pattern into three types—α-agarase, β-agarase, and β-porphyranase. Although, in a broad sense, many other agarases are involved in complete hydrolysis of agar, most of those identified are β-agarases. In this article we review agarolytic microorganisms and their agar-hydrolyzing systems, covering β-agarases as well as α-agarases, α-neoagarobiose hydrolases, and β-porphyranases, with emphasis on the recent discoveries. We also present an overview of the biochemical and structural characteristics of the various types of agarases. Further, we summarize and compare the agar-hydrolyzing systems of two specific microorganisms: Gram-negative Saccharophagus degradans 2–40 and Gram-positive Streptomyces coelicolor A3(2). We conclude with a brief discussion of the importance of agarases and their possible future application in producing oligosaccharides with various nutraceutical activities and in sustainably generating stock chemicals for biorefinement and bioenergy.
Keywords: Agar; Agarose; Porphyran; Agarase; Porphyranase; Agar degradation
Enzyme-based glucose delivery as a high content screening tool in yeast-based whole-cell biocatalysis by T. Grimm; M. Grimm; R. Klat; A. Neubauer; M. Palela; P. Neubauer (pp. 931-937).
The influence of glucose release on growth and biotransformation of yeasts was examined by using the medium EnBase® Flo in shake flasks. The medium contains a polysaccharide acting as substrate, which is degraded to glucose by the addition of an enzyme. In the present paper, this medium was adapted for the cultivation of yeasts by increasing the complex components (booster) and the enzyme concentrations to guarantee a higher glucose release rate. Important changes were an increase of the complex component booster to 10–15% and an increased glucose release by increasing the enzyme content to 15 U L−1. The 20 yeasts investigated in the present work showed an improvement of growth and biomass production when cultivated with the EnBase medium in comparison to yeast extract dextrose (YED) medium. Values of optical densities (OD600) of approximately 40 AU (corresponding to over 60 g L−1 wet cell weight) were achieved for all 20 yeast strains tested. During the following screening of the yeasts in whole-cell biotransformation, an improvement of the conversion for 19 out of the 20 yeasts cultivated with the EnBase Flo medium could be observed. The biomass from the EnBase Flo cultivation showed a higher conversion activity in the reduction of 2-butanone to (R/S)-2-butanol. The enantioselectivity (ee) of 15 yeast strains showed an improvement by using the EnBase medium. The number of yeasts with an ee >97% increased from zero with YED to six with EnBase medium. Thus, the use of a glucose release cultivation strategy in the screening process for transformation approaches provides significant benefits compared to standard batch approaches.
Keywords: Fed-batch cultivation; Glucose release; Whole-cell biocatalysis; Yeast screening; Enantioselectivity
Enzyme-based glucose delivery as a high content screening tool in yeast-based whole-cell biocatalysis by T. Grimm; M. Grimm; R. Klat; A. Neubauer; M. Palela; P. Neubauer (pp. 931-937).
The influence of glucose release on growth and biotransformation of yeasts was examined by using the medium EnBase® Flo in shake flasks. The medium contains a polysaccharide acting as substrate, which is degraded to glucose by the addition of an enzyme. In the present paper, this medium was adapted for the cultivation of yeasts by increasing the complex components (booster) and the enzyme concentrations to guarantee a higher glucose release rate. Important changes were an increase of the complex component booster to 10–15% and an increased glucose release by increasing the enzyme content to 15 U L−1. The 20 yeasts investigated in the present work showed an improvement of growth and biomass production when cultivated with the EnBase medium in comparison to yeast extract dextrose (YED) medium. Values of optical densities (OD600) of approximately 40 AU (corresponding to over 60 g L−1 wet cell weight) were achieved for all 20 yeast strains tested. During the following screening of the yeasts in whole-cell biotransformation, an improvement of the conversion for 19 out of the 20 yeasts cultivated with the EnBase Flo medium could be observed. The biomass from the EnBase Flo cultivation showed a higher conversion activity in the reduction of 2-butanone to (R/S)-2-butanol. The enantioselectivity (ee) of 15 yeast strains showed an improvement by using the EnBase medium. The number of yeasts with an ee >97% increased from zero with YED to six with EnBase medium. Thus, the use of a glucose release cultivation strategy in the screening process for transformation approaches provides significant benefits compared to standard batch approaches.
Keywords: Fed-batch cultivation; Glucose release; Whole-cell biocatalysis; Yeast screening; Enantioselectivity
Effect of pretreatment of hydrothermally processed rice straw with laccase-displaying yeast on ethanol fermentation by Akihito Nakanishi; Jun Gu Bae; Kotaro Fukai; Naoki Tokumoto; Kouichi Kuroda; Jun Ogawa; Masato Nakatani; Sakayu Shimizu; Mitsuyoshi Ueda (pp. 939-948).
A gene encoding laccase I was identified and cloned from the white-rot fungus Trametes sp. Ha1. Laccase I contained 10 introns and an original secretion signal sequence. After laccase I without introns was prepared by overlapping polymerase chain reaction, it was inserted into expression vector pULD1 for yeast cell surface display. The oxidation activity of a laccase-I-displaying yeast as a whole-cell biocatalyst was examined with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and the constructed yeast showed a high oxidation activity. After the pretreatment of hydrothermally processed rice straw (HPRS) with laccase-I-displaying yeast with ABTS, fermentation was conducted with yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase with HPRS. Fermentation of HPRS treated with laccase-I-displaying yeast was performed with 1.21-fold higher activities than those of HPRS treated with control yeast. The results indicated that pretreatment with laccase-I-displaying yeast with ABTS was effective for direct fermentation of cellulosic materials by yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase.
Keywords: Biorefinery; Laccase; Cell surface engineering of yeast; Trametes sp.; Lignin
Effect of pretreatment of hydrothermally processed rice straw with laccase-displaying yeast on ethanol fermentation by Akihito Nakanishi; Jun Gu Bae; Kotaro Fukai; Naoki Tokumoto; Kouichi Kuroda; Jun Ogawa; Masato Nakatani; Sakayu Shimizu; Mitsuyoshi Ueda (pp. 939-948).
A gene encoding laccase I was identified and cloned from the white-rot fungus Trametes sp. Ha1. Laccase I contained 10 introns and an original secretion signal sequence. After laccase I without introns was prepared by overlapping polymerase chain reaction, it was inserted into expression vector pULD1 for yeast cell surface display. The oxidation activity of a laccase-I-displaying yeast as a whole-cell biocatalyst was examined with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and the constructed yeast showed a high oxidation activity. After the pretreatment of hydrothermally processed rice straw (HPRS) with laccase-I-displaying yeast with ABTS, fermentation was conducted with yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase with HPRS. Fermentation of HPRS treated with laccase-I-displaying yeast was performed with 1.21-fold higher activities than those of HPRS treated with control yeast. The results indicated that pretreatment with laccase-I-displaying yeast with ABTS was effective for direct fermentation of cellulosic materials by yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase.
Keywords: Biorefinery; Laccase; Cell surface engineering of yeast; Trametes sp.; Lignin
Extracellular production of lipoxygenase from Anabaena sp. PCC 7120 in Bacillus subtilis and its effect on wheat protein by Chong Zhang; Tingting Tao; Qi Ying; Dongliang Zhang; Fengxia Lu; Xiaomei Bie; Zhaoxin Lu (pp. 949-958).
In this study, the lipoxygenase (ana-LOX) gene from Anabaena sp. PCC 7120 was successful expressed and secreted in Bacillus subtilis. Under the control of the P43 promoter, with a signal peptide from the B. subtilis 168 nprB gene, and facilitated by the molecular chaperone PrsA, the production of the recombinant ana-LOX (ana-rLOX) reached 76 U/mL (171.9 μg/ml) in the supernatant. The purified ana-rLOX was investigated for its effect on dough protein. Ana-rLOX treatment decreased free sulfhydryl groups, increased glutenin macropolymer content, promoted the formation of covalent bonds between gluten protein, and affected protein crosslinking. The results indicated that large aggregates involving gliadin and glutenin were formed. The glutenin macropolymer played a role in the formation of the dough network structure through the exchange of thiol disulfide bonds and the formation of hydrogen or hydrophobic bonds with other proteins.
Keywords: Anabaena sp. PCC 7120; Recombinant lipoxygenase (ana-rLOX); Bacillus subtilis ; Dough protein
Extracellular production of lipoxygenase from Anabaena sp. PCC 7120 in Bacillus subtilis and its effect on wheat protein by Chong Zhang; Tingting Tao; Qi Ying; Dongliang Zhang; Fengxia Lu; Xiaomei Bie; Zhaoxin Lu (pp. 949-958).
In this study, the lipoxygenase (ana-LOX) gene from Anabaena sp. PCC 7120 was successful expressed and secreted in Bacillus subtilis. Under the control of the P43 promoter, with a signal peptide from the B. subtilis 168 nprB gene, and facilitated by the molecular chaperone PrsA, the production of the recombinant ana-LOX (ana-rLOX) reached 76 U/mL (171.9 μg/ml) in the supernatant. The purified ana-rLOX was investigated for its effect on dough protein. Ana-rLOX treatment decreased free sulfhydryl groups, increased glutenin macropolymer content, promoted the formation of covalent bonds between gluten protein, and affected protein crosslinking. The results indicated that large aggregates involving gliadin and glutenin were formed. The glutenin macropolymer played a role in the formation of the dough network structure through the exchange of thiol disulfide bonds and the formation of hydrogen or hydrophobic bonds with other proteins.
Keywords: Anabaena sp. PCC 7120; Recombinant lipoxygenase (ana-rLOX); Bacillus subtilis ; Dough protein
Fermentation of xylose to succinate by enhancement of ATP supply in metabolically engineered Escherichia coli by Rongming Liu; Liya Liang; Kequan Chen; Jiangfeng Ma; Min Jiang; Ping Wei; Pingkai Ouyang (pp. 959-968).
In Escherichia coli K12, succinate was not the dominant fermentation product from xylose. To reduce by-product formation and increase succinate accumulation, pyruvate formate lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, these mutations eliminated cell growth and xylose utilization. During anaerobic growth of bacteria, organic intermediates, such as pyruvate, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate level phosphorylation. In E. coli K12, conversion of xylose to pyruvate only yielded 0.67 net ATP per xylose during anaerobic fermentation. However, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose, which could meet the ATP needed for xylose metabolism. A pflB deletion strain cannot convert pyruvate to acetyl coenzyme A, the precursor for acetate and ethanol production, and could not produce the additional ATP. Thus, the double mutations eliminated cell growth and xylose utilization. To supply the sufficient ATPs, overexpression of ATP-forming phosphoenolpyruvate-carboxykinase from Bacillus subtilis 168 in an ldhA, pflB, and ppc deletion strain resulted in a significant increase in cell mass and succinate production. In addition, fermentation of corn stalk hydrolysate containing a high percentage of xylose and glucose produced a final succinate concentration of 11.13 g l−1 with a yield of 1.02 g g−1 total sugars during anaerobic fermentation.
Keywords: ATP; Escherichia coli K12; Succinate; Corn stalk hydrolysate
Fermentation of xylose to succinate by enhancement of ATP supply in metabolically engineered Escherichia coli by Rongming Liu; Liya Liang; Kequan Chen; Jiangfeng Ma; Min Jiang; Ping Wei; Pingkai Ouyang (pp. 959-968).
In Escherichia coli K12, succinate was not the dominant fermentation product from xylose. To reduce by-product formation and increase succinate accumulation, pyruvate formate lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, these mutations eliminated cell growth and xylose utilization. During anaerobic growth of bacteria, organic intermediates, such as pyruvate, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate level phosphorylation. In E. coli K12, conversion of xylose to pyruvate only yielded 0.67 net ATP per xylose during anaerobic fermentation. However, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose, which could meet the ATP needed for xylose metabolism. A pflB deletion strain cannot convert pyruvate to acetyl coenzyme A, the precursor for acetate and ethanol production, and could not produce the additional ATP. Thus, the double mutations eliminated cell growth and xylose utilization. To supply the sufficient ATPs, overexpression of ATP-forming phosphoenolpyruvate-carboxykinase from Bacillus subtilis 168 in an ldhA, pflB, and ppc deletion strain resulted in a significant increase in cell mass and succinate production. In addition, fermentation of corn stalk hydrolysate containing a high percentage of xylose and glucose produced a final succinate concentration of 11.13 g l−1 with a yield of 1.02 g g−1 total sugars during anaerobic fermentation.
Keywords: ATP; Escherichia coli K12; Succinate; Corn stalk hydrolysate
Asymmetric synthesis of (R)-1,3-butanediol from 4-hydroxy-2-butanone by a newly isolated strain Candida krusei ZJB-09162 by Ren-Chao Zheng; Zheng Ge; Zhao-Kuan Qiu; Yuan-Shan Wang; Yu-Guo Zheng (pp. 969-976).
Biocatalytic asymmetric preparation of (R)-1,3-butanediol has been attracting much attention in pharmaceuticals industry. A new ideal strain, ZJB-09162, which exhibited high reduction activity and excellent (R)-stereospecificity towards 4-hydroxy-2-butanone, has been successfully isolated from soil samples. Based on morphology, physiological tests (API 20 C AUX), and 5.8S-ITS sequence, the isolate was identified as Candida krusei. Kinetic characterization demonstrated that carbonyl reductase from C. krusei ZJB-09162 preferred NADH to NADPH as cofactor, indicating it might be a new carbonyl reductase. (R)-1,3-Butanediol was produced in 19.8 g/L, 96.6% conversion, and 99.0% ee at optimal pH 8.5, 35 °C with a 2:1 molar ratio of glucose to 4H2B. In order to achieve higher product titer, the substrate loading was optimized in fixed catalysts and fixed substrate/catalysts ratio mode. The bioreduction of 4-hydroxy-2-butanone at a concentration of 45.0 g/L gave (R)-1,3-butanediol in 38.7 g/L and 83.9% conversion. Therefore, C. krusei ZJB-09162 was, for the first time, proven to be a promising biocatalyst for enzymatic preparation of (R)-1,3-butanediol.
Keywords: (R)-1,3-Butanediol; Candida krusei ZJB-09162; Carbonyl reductase; Asymmetric reduction; NADH-dependent
Asymmetric synthesis of (R)-1,3-butanediol from 4-hydroxy-2-butanone by a newly isolated strain Candida krusei ZJB-09162 by Ren-Chao Zheng; Zheng Ge; Zhao-Kuan Qiu; Yuan-Shan Wang; Yu-Guo Zheng (pp. 969-976).
Biocatalytic asymmetric preparation of (R)-1,3-butanediol has been attracting much attention in pharmaceuticals industry. A new ideal strain, ZJB-09162, which exhibited high reduction activity and excellent (R)-stereospecificity towards 4-hydroxy-2-butanone, has been successfully isolated from soil samples. Based on morphology, physiological tests (API 20 C AUX), and 5.8S-ITS sequence, the isolate was identified as Candida krusei. Kinetic characterization demonstrated that carbonyl reductase from C. krusei ZJB-09162 preferred NADH to NADPH as cofactor, indicating it might be a new carbonyl reductase. (R)-1,3-Butanediol was produced in 19.8 g/L, 96.6% conversion, and 99.0% ee at optimal pH 8.5, 35 °C with a 2:1 molar ratio of glucose to 4H2B. In order to achieve higher product titer, the substrate loading was optimized in fixed catalysts and fixed substrate/catalysts ratio mode. The bioreduction of 4-hydroxy-2-butanone at a concentration of 45.0 g/L gave (R)-1,3-butanediol in 38.7 g/L and 83.9% conversion. Therefore, C. krusei ZJB-09162 was, for the first time, proven to be a promising biocatalyst for enzymatic preparation of (R)-1,3-butanediol.
Keywords: (R)-1,3-Butanediol; Candida krusei ZJB-09162; Carbonyl reductase; Asymmetric reduction; NADH-dependent
PmST3 from Pasteurella multocida encoded by Pm1174 gene is a monofunctional α2–3-sialyltransferase by Vireak Thon; Yanhong Li; Hai Yu; Kam Lau; Xi Chen (pp. 977-985).
Pasteurella multocida (Pm) strain Pm70 has three putative sialyltransferase genes including Pm0188, Pm0508, and Pm1174. A Pm0188 gene homolog in Pm strain P-1059 encodes a multifunctional α2–3-sialyltransferase, PmST1, that prefers oligosaccharide acceptors. A Pm0508 gene homolog in the same strain encodes a monofunctional sialyltransferase PmST2 that prefers glycolipid acceptors. Here, we report that the third sialyltransferase from Pm (PmST3) encoded by gene Pm1174 in strain Pm70 is a monofunctional α2–3-sialyltransferase that can use both oligosaccharides and glycolipids as efficient acceptors. Despite the existence of both Pm0188 and Pm0508 gene homologs encoding PmST1 and PmST2, respectively, in Pm strain P-1059, a Pm1174 gene homolog appears to be absent from Pm strains P-1059 and P-934. PmST3 was successfully obtained by cloning and expression using a synthetic gene of Pm1174 with codons optimized for Escherichia coli expression system as the DNA template for polymer chain reactions. Truncation of 35 amino acid residues from the carboxyl terminus was shown to improve the expression of a soluble and active enzyme in E. coli as a C-His6-tagged fusion protein. This sialidase-free monofunctional α2–3-sialyltransferase is a useful tool for synthesizing sialylated oligosaccharides and glycolipids.
Keywords: Carbohydrate synthesis; Glycosyltransferase; Pasteurella multocida ; Pasteurella multocida sialyltransferase 3; Sialic acid; Sialyltransferase
PmST3 from Pasteurella multocida encoded by Pm1174 gene is a monofunctional α2–3-sialyltransferase by Vireak Thon; Yanhong Li; Hai Yu; Kam Lau; Xi Chen (pp. 977-985).
Pasteurella multocida (Pm) strain Pm70 has three putative sialyltransferase genes including Pm0188, Pm0508, and Pm1174. A Pm0188 gene homolog in Pm strain P-1059 encodes a multifunctional α2–3-sialyltransferase, PmST1, that prefers oligosaccharide acceptors. A Pm0508 gene homolog in the same strain encodes a monofunctional sialyltransferase PmST2 that prefers glycolipid acceptors. Here, we report that the third sialyltransferase from Pm (PmST3) encoded by gene Pm1174 in strain Pm70 is a monofunctional α2–3-sialyltransferase that can use both oligosaccharides and glycolipids as efficient acceptors. Despite the existence of both Pm0188 and Pm0508 gene homologs encoding PmST1 and PmST2, respectively, in Pm strain P-1059, a Pm1174 gene homolog appears to be absent from Pm strains P-1059 and P-934. PmST3 was successfully obtained by cloning and expression using a synthetic gene of Pm1174 with codons optimized for Escherichia coli expression system as the DNA template for polymer chain reactions. Truncation of 35 amino acid residues from the carboxyl terminus was shown to improve the expression of a soluble and active enzyme in E. coli as a C-His6-tagged fusion protein. This sialidase-free monofunctional α2–3-sialyltransferase is a useful tool for synthesizing sialylated oligosaccharides and glycolipids.
Keywords: Carbohydrate synthesis; Glycosyltransferase; Pasteurella multocida ; Pasteurella multocida sialyltransferase 3; Sialic acid; Sialyltransferase
A hemolytic peptide from the mycophilic fungus Sepedonium chrysospermum (Bull.) Fr. by Elisa Sanguineti; Maria E. Cosulich; Annalisa Salis; Gianluca Damonte; Mauro G. Mariotti; Mirca Zotti (pp. 987-994).
The hemolytic activity of an extract of the mycoparasite Sepedonium chrysospermum (teleomorph Hypomyces chrysospermus) was detected and characterized. Extraction of the fungal biomass by methanol yielded a fraction in which the hemolytic activity against human red blood cells corresponded to a peptide with a molecular mass of 7,653.72 Da and an isoelectric point of approximately 5.8. The peptide was temperature resistant, and the hemolysis was only partially inhibited, even after a 30-min pre-incubation at 100°C. Its hemolytic activity was unaffected by treatment with proteolytic enzymes such as trypsin. Among the divalent cations assayed, Hg2+ was the strongest inhibitor of hemolysis. The reducing agent, dithiothreitol, and the membrane lipid, cholesterol, demonstrated concentration-dependent inhibitory activities. Finally, hemolytic activity triggered by the peptide was analyzed by scanning electron microscopy, and a pore-forming activity was detected.
Keywords: Mycoparasite; Hemolysis; Sepedonium chrysospermum ; Peptide
A hemolytic peptide from the mycophilic fungus Sepedonium chrysospermum (Bull.) Fr. by Elisa Sanguineti; Maria E. Cosulich; Annalisa Salis; Gianluca Damonte; Mauro G. Mariotti; Mirca Zotti (pp. 987-994).
The hemolytic activity of an extract of the mycoparasite Sepedonium chrysospermum (teleomorph Hypomyces chrysospermus) was detected and characterized. Extraction of the fungal biomass by methanol yielded a fraction in which the hemolytic activity against human red blood cells corresponded to a peptide with a molecular mass of 7,653.72 Da and an isoelectric point of approximately 5.8. The peptide was temperature resistant, and the hemolysis was only partially inhibited, even after a 30-min pre-incubation at 100°C. Its hemolytic activity was unaffected by treatment with proteolytic enzymes such as trypsin. Among the divalent cations assayed, Hg2+ was the strongest inhibitor of hemolysis. The reducing agent, dithiothreitol, and the membrane lipid, cholesterol, demonstrated concentration-dependent inhibitory activities. Finally, hemolytic activity triggered by the peptide was analyzed by scanning electron microscopy, and a pore-forming activity was detected.
Keywords: Mycoparasite; Hemolysis; Sepedonium chrysospermum ; Peptide
A family GH51 α-l-arabinofuranosidase from Pleurotus ostreatus: identification, recombinant expression and characterization by Antonella Amore; Angela Amoresano; Leila Birolo; Bernard Henrissat; Gabriella Leo; Angelo Palmese; Vincenza Faraco (pp. 995-1006).
An α-l-arabinofuranosidase produced by Pleurotus ostreatus (PoAbf) during solid state fermentation on tomato pomace was identified and the corresponding gene and cDNA were cloned and sequenced. Molecular analysis showed that the poabf gene carries 26 exons interrupted by 25 introns and has an open reading frame encoding a protein of 646 amino acid residues, including a signal peptide of 20 amino acid residues. The amino acid sequence similar to the other α-l-arabinofuranosidases indicated that the enzyme encoded by poabf can be classified as a family 51 glycoside hydrolase. Heterologous recombinant expression of PoAbf was carried out in the yeasts Pichia pastoris and Kluyveromyces lactis achieving the highest production level of the secreted enzyme (180 mg L−1) in the former host. rPoAbf produced in P. pastoris was purified and characterized. It is a glycosylated monomer with a molecular weight of 81,500 Da in denaturing conditions. Mass spectral analyses led to the localization of a single O-glycosylation site at the level of Ser160. The enzyme is highly specific for α-l-arabinofuranosyl linkages and when assayed with p-nitrophenyl α-l-arabinofuranoside it follows Michaelis–Menten kinetics with a K M of 0.64 mM and a k cat of 3,010 min−1. The optimum pH is 5 and the optimal temperature 40°C. It is worth noting that the enzyme shows a very high stability in a broad range of pH. The more durable activity showed by rPoAbf in comparison to the other α-l-arabinofuranosidases enhances its potential for biotechnological applications and increases interest in elucidating the molecular bases of its peculiar properties.
Keywords: α-l-arabinofuranosidase; Fungus; Recombinant expression; Pichia pastoris
A family GH51 α-l-arabinofuranosidase from Pleurotus ostreatus: identification, recombinant expression and characterization by Antonella Amore; Angela Amoresano; Leila Birolo; Bernard Henrissat; Gabriella Leo; Angelo Palmese; Vincenza Faraco (pp. 995-1006).
An α-l-arabinofuranosidase produced by Pleurotus ostreatus (PoAbf) during solid state fermentation on tomato pomace was identified and the corresponding gene and cDNA were cloned and sequenced. Molecular analysis showed that the poabf gene carries 26 exons interrupted by 25 introns and has an open reading frame encoding a protein of 646 amino acid residues, including a signal peptide of 20 amino acid residues. The amino acid sequence similar to the other α-l-arabinofuranosidases indicated that the enzyme encoded by poabf can be classified as a family 51 glycoside hydrolase. Heterologous recombinant expression of PoAbf was carried out in the yeasts Pichia pastoris and Kluyveromyces lactis achieving the highest production level of the secreted enzyme (180 mg L−1) in the former host. rPoAbf produced in P. pastoris was purified and characterized. It is a glycosylated monomer with a molecular weight of 81,500 Da in denaturing conditions. Mass spectral analyses led to the localization of a single O-glycosylation site at the level of Ser160. The enzyme is highly specific for α-l-arabinofuranosyl linkages and when assayed with p-nitrophenyl α-l-arabinofuranoside it follows Michaelis–Menten kinetics with a K M of 0.64 mM and a k cat of 3,010 min−1. The optimum pH is 5 and the optimal temperature 40°C. It is worth noting that the enzyme shows a very high stability in a broad range of pH. The more durable activity showed by rPoAbf in comparison to the other α-l-arabinofuranosidases enhances its potential for biotechnological applications and increases interest in elucidating the molecular bases of its peculiar properties.
Keywords: α-l-arabinofuranosidase; Fungus; Recombinant expression; Pichia pastoris
Cloning and characterization of a novel amidase from Paracoccus sp. M-1, showing aryl acylamidase and acyl transferase activities by Weiliang Shen; Honghong Chen; Kaizhi Jia; Jun Ni; Xin Yan; Shunpeng Li (pp. 1007-1018).
A novel amidase gene, designated pamh, was cloned from Paracoccus sp. M-1. Site-directed mutagenesis and bioinformatic analysis showed that the PamH protein belonged to the amidase signature enzyme family. PamH was expressed in Escherichia coli, purified, and characterized. The molecular mass of PamH was determined to be 52 kDa with an isoelectric point of 5.13. PamH displayed its highest enzymatic activity at 45°C and at pH 8.0 and was stable within a pH range of 5.0–10.0. The PamH enzyme exhibited amidase activity, aryl acylamidase activity, and acyl transferase activity, allowing it to function across a very broad substrate spectrum. PamH was highly active on aromatic and short-chain aliphatic amides (benzamide and propionamide), moderately active on amino acid amides, and possessed weak urease activity. Of the anilides examined, only propanil was a good substrate for PamH. For propanil, the k cat and K m were 2.8 s−1 and 158 μM, respectively, and the catalytic efficiency value (k cat/K m) was 0.018 μM−1 s−1. In addition, PamH was able to catalyze the acyl transfer reaction to hydroxylamine for both amide and anilide substrates, including acetamide, propanil, and 4-nitroacetanilide; the highest reaction rate was shown with isobutyramide. These characteristics make PamH an excellent candidate for environmental remediation and an important enzyme for the biosynthesis of novel amides.
Keywords: PamH; Amidase; Aryl acylamidase; Acyl transferase activity; Propanil; Gene cloning
Cloning and characterization of a novel amidase from Paracoccus sp. M-1, showing aryl acylamidase and acyl transferase activities by Weiliang Shen; Honghong Chen; Kaizhi Jia; Jun Ni; Xin Yan; Shunpeng Li (pp. 1007-1018).
A novel amidase gene, designated pamh, was cloned from Paracoccus sp. M-1. Site-directed mutagenesis and bioinformatic analysis showed that the PamH protein belonged to the amidase signature enzyme family. PamH was expressed in Escherichia coli, purified, and characterized. The molecular mass of PamH was determined to be 52 kDa with an isoelectric point of 5.13. PamH displayed its highest enzymatic activity at 45°C and at pH 8.0 and was stable within a pH range of 5.0–10.0. The PamH enzyme exhibited amidase activity, aryl acylamidase activity, and acyl transferase activity, allowing it to function across a very broad substrate spectrum. PamH was highly active on aromatic and short-chain aliphatic amides (benzamide and propionamide), moderately active on amino acid amides, and possessed weak urease activity. Of the anilides examined, only propanil was a good substrate for PamH. For propanil, the k cat and K m were 2.8 s−1 and 158 μM, respectively, and the catalytic efficiency value (k cat/K m) was 0.018 μM−1 s−1. In addition, PamH was able to catalyze the acyl transfer reaction to hydroxylamine for both amide and anilide substrates, including acetamide, propanil, and 4-nitroacetanilide; the highest reaction rate was shown with isobutyramide. These characteristics make PamH an excellent candidate for environmental remediation and an important enzyme for the biosynthesis of novel amides.
Keywords: PamH; Amidase; Aryl acylamidase; Acyl transferase activity; Propanil; Gene cloning
Engineering of LadA for enhanced hexadecane oxidation using random- and site-directed mutagenesis by Yanpeng Dong; Jiang Yan; Huiqian Du; Miao Chen; Ting Ma; Lu Feng (pp. 1019-1029).
LadA, a monooxygenase catalyzing the oxidation of n-alkanes to 1-alkanols, is the key enzyme for the degradation of long-chain alkanes (C15–C36) in Geobacillus thermodenitrificans NG80-2. In this study, random- and site-directed mutagenesis were performed to enhance the activity of the enzyme. By screening 7,500 clones from random-mutant libraries for enhanced hexadecane hydroxylation activity, three mutants were obtained: A102D, L320V, and F146C/N376I. By performing saturation site-directed mutagenesis at the 102, 320, 146, and 376 sites, six more mutants (A102E, L320A, F146Q/N376I, F146E/N376I, F146R/N376I, and F146N/N376I) were generated. Kinetic studies showed that the hydroxylation activity of purified LadA mutants on hexadecane was 2–3.4-fold higher than that of the wild-type enzyme, with the activity of F146N/N376I being the highest. Effects of the mutations on optimum temperature, pH, and heat stability of LadA were also investigated. A complementary study showed that Pseudomonas fluorescens KOB2Δ1 strains expressing the LadA mutants grew more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutants in vivo. Structural changes resulting from the mutations were analyzed and the correlation between structural changes and enzyme activity was discussed. The mutants generated in this study are potentially useful for the treatment of environmental oil pollution and in other bioconversion processes.
Keywords: LadA; Monooxygenase; Hydroxylase; Directed evolution; Error-prone PCR; Saturation site-directed mutagenesis; Hexadecane
Engineering of LadA for enhanced hexadecane oxidation using random- and site-directed mutagenesis by Yanpeng Dong; Jiang Yan; Huiqian Du; Miao Chen; Ting Ma; Lu Feng (pp. 1019-1029).
LadA, a monooxygenase catalyzing the oxidation of n-alkanes to 1-alkanols, is the key enzyme for the degradation of long-chain alkanes (C15–C36) in Geobacillus thermodenitrificans NG80-2. In this study, random- and site-directed mutagenesis were performed to enhance the activity of the enzyme. By screening 7,500 clones from random-mutant libraries for enhanced hexadecane hydroxylation activity, three mutants were obtained: A102D, L320V, and F146C/N376I. By performing saturation site-directed mutagenesis at the 102, 320, 146, and 376 sites, six more mutants (A102E, L320A, F146Q/N376I, F146E/N376I, F146R/N376I, and F146N/N376I) were generated. Kinetic studies showed that the hydroxylation activity of purified LadA mutants on hexadecane was 2–3.4-fold higher than that of the wild-type enzyme, with the activity of F146N/N376I being the highest. Effects of the mutations on optimum temperature, pH, and heat stability of LadA were also investigated. A complementary study showed that Pseudomonas fluorescens KOB2Δ1 strains expressing the LadA mutants grew more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutants in vivo. Structural changes resulting from the mutations were analyzed and the correlation between structural changes and enzyme activity was discussed. The mutants generated in this study are potentially useful for the treatment of environmental oil pollution and in other bioconversion processes.
Keywords: LadA; Monooxygenase; Hydroxylase; Directed evolution; Error-prone PCR; Saturation site-directed mutagenesis; Hexadecane
Production, purification, and characterization of the cecropin from Plutella xylostella, pxCECA1, using an intein-induced self-cleavable system in Escherichia coli by Hong Wang; Xiao-lin Meng; Jin-ping Xu; Jian Wang; Hua Wang; Chun-wei Ma (pp. 1031-1039).
Antimicrobial peptides (AMPs) are widely expressed and play an important role in innate immune defense against infectious agents such as bacteria, viruses, fungi, and parasites. Cecropins are a family of AMPs synthesized in the fat body of insects that have proven effective at killing specific pathogens. In order to fulfill their clinical potential as antimicrobial drugs, a simple, cost-effective method to express AMPs is sorely needed. In this study, we expressed and characterized the cecropin from Plutella xylostella (pxCECA1) using an intein-dependent expression system in Escherichia coli. We cloned the pxCECA1 gene from larva by RT-PCR and fused the encoding sequence of mature pxCECA1 with an intein gene and a chitin-binding domain gene (CBD) in pTWIN1 plasmid. The fusion protein CBD–intein–pxCECA1 was expressed in E. coli BL21 (DE3) and separated by flowing cell extracts through a chitin column. Subsequently, self-cleavage of the intein at its C-terminus was induced in a temperature- and pH-dependent manner, resulting in the release of mature pxCECA1. The optimal conditions for self-cleavage were determined to be pH 6.0 for 48 h at 4°C, under which 12.3 mg of recombinant pxCECA1 could be recovered from 1 l of E. coli culture. The purified pxCECA1 displayed antimicrobial activity against a broad variety of gram-positive and gram-negative bacteria. This preparation was especially effective against Staphylococcus aureus, including methicillin-resistant strains. Catalase release assays demonstrated that pxCECA1 acts as a microbicidal agent. These results show for the first time that the IMPACT-TWIN expression system is an efficient, cost-effective way to produce fully functional AMPs and that the AMP pxCECA1 is a novel microbicidal agent with promising therapeutic applications.
Keywords: Plutella xylostella ; Cecropin; Self-cleavage; Antimicrobial activity
Production, purification, and characterization of the cecropin from Plutella xylostella, pxCECA1, using an intein-induced self-cleavable system in Escherichia coli by Hong Wang; Xiao-lin Meng; Jin-ping Xu; Jian Wang; Hua Wang; Chun-wei Ma (pp. 1031-1039).
Antimicrobial peptides (AMPs) are widely expressed and play an important role in innate immune defense against infectious agents such as bacteria, viruses, fungi, and parasites. Cecropins are a family of AMPs synthesized in the fat body of insects that have proven effective at killing specific pathogens. In order to fulfill their clinical potential as antimicrobial drugs, a simple, cost-effective method to express AMPs is sorely needed. In this study, we expressed and characterized the cecropin from Plutella xylostella (pxCECA1) using an intein-dependent expression system in Escherichia coli. We cloned the pxCECA1 gene from larva by RT-PCR and fused the encoding sequence of mature pxCECA1 with an intein gene and a chitin-binding domain gene (CBD) in pTWIN1 plasmid. The fusion protein CBD–intein–pxCECA1 was expressed in E. coli BL21 (DE3) and separated by flowing cell extracts through a chitin column. Subsequently, self-cleavage of the intein at its C-terminus was induced in a temperature- and pH-dependent manner, resulting in the release of mature pxCECA1. The optimal conditions for self-cleavage were determined to be pH 6.0 for 48 h at 4°C, under which 12.3 mg of recombinant pxCECA1 could be recovered from 1 l of E. coli culture. The purified pxCECA1 displayed antimicrobial activity against a broad variety of gram-positive and gram-negative bacteria. This preparation was especially effective against Staphylococcus aureus, including methicillin-resistant strains. Catalase release assays demonstrated that pxCECA1 acts as a microbicidal agent. These results show for the first time that the IMPACT-TWIN expression system is an efficient, cost-effective way to produce fully functional AMPs and that the AMP pxCECA1 is a novel microbicidal agent with promising therapeutic applications.
Keywords: Plutella xylostella ; Cecropin; Self-cleavage; Antimicrobial activity
Inhibition of anthrax lethal factor: lability of hydroxamate as a chelating group by Feng Li; Irina Chvyrkova; Simon Terzyan; Nancy Wakeham; Robert Turner; Arun K. Ghosh; Xuejun C. Zhang; Jordan Tang (pp. 1041-1049).
The metalloprotease activity of lethal factor (LF) from Bacillus anthracis (B. anthracis) is a main source of toxicity in the lethality of anthrax infection. Thus, the understanding of the enzymatic activity and inhibition of B. anthracis LF is of scientific and clinical interests. We have designed, synthesized, and studied a peptide inhibitor of LF, R9LF-1, with the structure NH2–(d-Arg)9–Val–Leu–Arg–CO–NHOH in which the C-terminal hydroxamic acid is commonly used in the inhibitors of metalloproteases to chelate the active-site zinc. This inhibitor was shown to be very stable in solution and effectively inhibited LF in kinetic assays. However, its protection on murine macrophages against lethal toxin’s lysis activity was relatively weak in longer assays. We further observed that the hydroxamic acid group in R9LF-1 was hydrolyzed by LF, and the hydrolytic product of this inhibitor is considerably weaker in inhibition of potency. To resist this unique hydrolytic activity of LF, we further designed a new inhibitor R9LF-2 which contained the same structure as R9LF-1 except replacing the hydroxamic acid group with N,O-dimethyl hydroxamic acid (DMHA), –N(CH3)–O–CH3. R9LF-2 was not hydrolyzed by LF in long-term incubation. It has a high inhibitory potency vs. LF with an inhibition constant of 6.4 nM had a better protection of macrophages against LF toxicity than R9LF-1. These results suggest that in the development of new LF inhibitors, the stability of the chelating group should be carefully examined and that DMHA is a potentially useful moiety to be used in new LF inhibitors.
Keywords: Anthrax; Lethal toxin; Inhibitor; Hydroxamate; Metalloprotease
Inhibition of anthrax lethal factor: lability of hydroxamate as a chelating group by Feng Li; Irina Chvyrkova; Simon Terzyan; Nancy Wakeham; Robert Turner; Arun K. Ghosh; Xuejun C. Zhang; Jordan Tang (pp. 1041-1049).
The metalloprotease activity of lethal factor (LF) from Bacillus anthracis (B. anthracis) is a main source of toxicity in the lethality of anthrax infection. Thus, the understanding of the enzymatic activity and inhibition of B. anthracis LF is of scientific and clinical interests. We have designed, synthesized, and studied a peptide inhibitor of LF, R9LF-1, with the structure NH2–(d-Arg)9–Val–Leu–Arg–CO–NHOH in which the C-terminal hydroxamic acid is commonly used in the inhibitors of metalloproteases to chelate the active-site zinc. This inhibitor was shown to be very stable in solution and effectively inhibited LF in kinetic assays. However, its protection on murine macrophages against lethal toxin’s lysis activity was relatively weak in longer assays. We further observed that the hydroxamic acid group in R9LF-1 was hydrolyzed by LF, and the hydrolytic product of this inhibitor is considerably weaker in inhibition of potency. To resist this unique hydrolytic activity of LF, we further designed a new inhibitor R9LF-2 which contained the same structure as R9LF-1 except replacing the hydroxamic acid group with N,O-dimethyl hydroxamic acid (DMHA), –N(CH3)–O–CH3. R9LF-2 was not hydrolyzed by LF in long-term incubation. It has a high inhibitory potency vs. LF with an inhibition constant of 6.4 nM had a better protection of macrophages against LF toxicity than R9LF-1. These results suggest that in the development of new LF inhibitors, the stability of the chelating group should be carefully examined and that DMHA is a potentially useful moiety to be used in new LF inhibitors.
Keywords: Anthrax; Lethal toxin; Inhibitor; Hydroxamate; Metalloprotease
Effective expression of soluble aglycosylated recombinant human Fcγ receptor I by low translational efficiency in Escherichia coli by Kouta Hatayama; Yoshiharu Asaoka; Megumi Hoya; Teruhiko Ide (pp. 1051-1059).
Human FcγRI (CD64) is an integral membrane glycoprotein functioning as a high-affinity receptor binding to monomeric IgG. In this study, the extracellular region of FcγRI, which is the actual part that interacts with IgG, was expressed as aglycosylated recombinant human FcγRI (rhFcγRI) in Escherichia coli. The soluble form of aglycosylated rhFcγRI was expressed in the periplasm of E. coli. The production of soluble aglycosylated rhFcγRI was increased by low induction levels. Furthermore, this production was increased by low translational efficiency, controlled by modification of the putative region between the ribosome binding site and initiation codon of rhFcγRI fusing signal peptide (MalE, PelB, or TorT) of the expression vector. By the optimization of induction and translational efficiency, the production of soluble aglycosylated rhFcγRI was up to approximately 0.8 mg/l of culture medium. Surface plasmon resonance analysis revealed that the binding affinities of aglycosylated rhFcγRI for human IgG1 (equilibrium dissociation constant K D = [1.7 ± 0.2] × 10−10 M) and IgG3 (K D = [1.1 ± 0.2] × 10−10 M) were similar to those of glycosylated rhFcγRI.
Keywords: FcγRI; CD64; Aglycosylated; Recombinant; Translational efficiency; Binding affinity
Effective expression of soluble aglycosylated recombinant human Fcγ receptor I by low translational efficiency in Escherichia coli by Kouta Hatayama; Yoshiharu Asaoka; Megumi Hoya; Teruhiko Ide (pp. 1051-1059).
Human FcγRI (CD64) is an integral membrane glycoprotein functioning as a high-affinity receptor binding to monomeric IgG. In this study, the extracellular region of FcγRI, which is the actual part that interacts with IgG, was expressed as aglycosylated recombinant human FcγRI (rhFcγRI) in Escherichia coli. The soluble form of aglycosylated rhFcγRI was expressed in the periplasm of E. coli. The production of soluble aglycosylated rhFcγRI was increased by low induction levels. Furthermore, this production was increased by low translational efficiency, controlled by modification of the putative region between the ribosome binding site and initiation codon of rhFcγRI fusing signal peptide (MalE, PelB, or TorT) of the expression vector. By the optimization of induction and translational efficiency, the production of soluble aglycosylated rhFcγRI was up to approximately 0.8 mg/l of culture medium. Surface plasmon resonance analysis revealed that the binding affinities of aglycosylated rhFcγRI for human IgG1 (equilibrium dissociation constant K D = [1.7 ± 0.2] × 10−10 M) and IgG3 (K D = [1.1 ± 0.2] × 10−10 M) were similar to those of glycosylated rhFcγRI.
Keywords: FcγRI; CD64; Aglycosylated; Recombinant; Translational efficiency; Binding affinity
Cloning and heterologous expression of a bacteriocin sakacin P from Lactobacillus sakei in Escherichia coli by Haiqin Chen; Fengwei Tian; Shuo Li; Yan Xie; Hao Zhang; Wei Chen (pp. 1061-1068).
Sakacin P, a bacteriocin from Lactobacillus sakei, shows strong activity against food-borne pathogens such as Listeria monocytogenes. In L. sakei, the structural gene (sppA) encoding sakacin P is controlled by a strict regulatory mechanism, and the quantity of secreted sakacin P is limited. In this study, the sppA gene was synthesized by splicing overlap extension PCR and cloned into Escherichia coli. After the induction with isopropyl-β-d-thiogalactopyranoside, the recombinant sakacin P was successfully expressed. The collected cells were sonicated, and the activity was detected by agar diffusion method. The results also showed that the low-temperature induction can improve the activity of sakacin P.
Keywords: Sakacin P; Expression; Class IIa bacteriocins
Cloning and heterologous expression of a bacteriocin sakacin P from Lactobacillus sakei in Escherichia coli by Haiqin Chen; Fengwei Tian; Shuo Li; Yan Xie; Hao Zhang; Wei Chen (pp. 1061-1068).
Sakacin P, a bacteriocin from Lactobacillus sakei, shows strong activity against food-borne pathogens such as Listeria monocytogenes. In L. sakei, the structural gene (sppA) encoding sakacin P is controlled by a strict regulatory mechanism, and the quantity of secreted sakacin P is limited. In this study, the sppA gene was synthesized by splicing overlap extension PCR and cloned into Escherichia coli. After the induction with isopropyl-β-d-thiogalactopyranoside, the recombinant sakacin P was successfully expressed. The collected cells were sonicated, and the activity was detected by agar diffusion method. The results also showed that the low-temperature induction can improve the activity of sakacin P.
Keywords: Sakacin P; Expression; Class IIa bacteriocins
Evolution and some functions of the NprR–NprRB quorum-sensing system in the Bacillus cereus group by Jorge Rocha; Victor Flores; Rosina Cabrera; Adriana Soto-Guzmán; Giovana Granados; Eusebio Juaristi; Gabriel Guarneros; Mayra de la Torre (pp. 1069-1078).
Quorum-sensing (QS) is a bacterial mechanism for regulation of gene expression in response to cell density. In Gram-positive bacteria, oligopeptides are the signaling molecules to elicit QS. The RNPP protein family (Rap, NprR, PlcR, and PrgX) are intracellular QS receptors that bind directly to their specific signaling peptide for regulating the transcription of several genes. NprR is the activator of a neutral protease in Bacillus subtilis, and it has been recently related to sporulation, cry genes transcription and extracellular protease activity in strains from the B. cereus group. In the B. thuringiensis genome, downstream nprR, a gene encoding a putative QS signaling propeptide (nprRB) was found. We hypothesized that the nprR and nprRB co-evolved because of their coordinated function in the B. cereus group. A phylogenetic tree of nucleotide sequences of nprR revealed six pherotypes, each corresponding to one putative mature NprRB sequence. The nprR tree does not match the current taxonomic grouping of the B. cereus group or the phylogenetic arrangement obtained when using MLST markers from the same strains. SKPDI and other synthetic peptides encoded in the nprRB gene from B. thuringiensis serovar thuringiensis strain 8741 had effect on temporal regulation of sporulation and expression of a cry1Aa’Z transcriptional fusion, but those peptides that stimulated earlier detection of spores decreased cry1Aa expression suggesting that NprR may either activate or repress the transcription of different genes.
Keywords: Bacillus thuringiensis ; Quorum-sensing; Sporulation-regulation; cry1Aa expression
Evolution and some functions of the NprR–NprRB quorum-sensing system in the Bacillus cereus group by Jorge Rocha; Victor Flores; Rosina Cabrera; Adriana Soto-Guzmán; Giovana Granados; Eusebio Juaristi; Gabriel Guarneros; Mayra de la Torre (pp. 1069-1078).
Quorum-sensing (QS) is a bacterial mechanism for regulation of gene expression in response to cell density. In Gram-positive bacteria, oligopeptides are the signaling molecules to elicit QS. The RNPP protein family (Rap, NprR, PlcR, and PrgX) are intracellular QS receptors that bind directly to their specific signaling peptide for regulating the transcription of several genes. NprR is the activator of a neutral protease in Bacillus subtilis, and it has been recently related to sporulation, cry genes transcription and extracellular protease activity in strains from the B. cereus group. In the B. thuringiensis genome, downstream nprR, a gene encoding a putative QS signaling propeptide (nprRB) was found. We hypothesized that the nprR and nprRB co-evolved because of their coordinated function in the B. cereus group. A phylogenetic tree of nucleotide sequences of nprR revealed six pherotypes, each corresponding to one putative mature NprRB sequence. The nprR tree does not match the current taxonomic grouping of the B. cereus group or the phylogenetic arrangement obtained when using MLST markers from the same strains. SKPDI and other synthetic peptides encoded in the nprRB gene from B. thuringiensis serovar thuringiensis strain 8741 had effect on temporal regulation of sporulation and expression of a cry1Aa’Z transcriptional fusion, but those peptides that stimulated earlier detection of spores decreased cry1Aa expression suggesting that NprR may either activate or repress the transcription of different genes.
Keywords: Bacillus thuringiensis ; Quorum-sensing; Sporulation-regulation; cry1Aa expression
An engineered Escherichia coli having a high intracellular level of ATP and enhanced recombinant protein production by Hye-Jung Kim; Yeong Deok Kwon; Sang Yup Lee; Pil Kim (pp. 1079-1086).
Artificial amplification of gluconeogenic phosphoenolpyruvate carboxykinase (PCK) under glycolytic conditions enables Escherichia coli to maintain a greater intracellular ATP concentration during its growth phase. To demonstrate the biotechnological benefit of E. coli harboring a high intracellular ATP concentration, we compared the recombinant protein synthesis of a soluble protein (enhanced green fluorescence protein, GFP) with that of a secretory protein (alkaline protease, AP), under control of the T7 promoter in E. coli BL21(DE3) overexpressing PCK. According to the batch fermentations, the strain overexpressing PCK produced more GFP and AP with a lower increase in biomass than the control strain. In a chemostat culture (D = 0.7 h−1), the GFP production in the PCK overexpressing strain was 99.0 ± 4.31 mg/g cell, with a biomass of 0.22 g/L, while that of the control strain was 53.5 ± 3.07 mg/g cell, with a biomass of 0.35 g/L. These results indicate that the PCK overexpressing E. coli strain harboring high intracellular levels of ATP can be useful as a protein-synthesizing host. The potential uses of the strain and associated rationale are discussed.
Keywords: High intracellular ATP concentration; Recombinant protein expression; Cellular energy
An engineered Escherichia coli having a high intracellular level of ATP and enhanced recombinant protein production by Hye-Jung Kim; Yeong Deok Kwon; Sang Yup Lee; Pil Kim (pp. 1079-1086).
Artificial amplification of gluconeogenic phosphoenolpyruvate carboxykinase (PCK) under glycolytic conditions enables Escherichia coli to maintain a greater intracellular ATP concentration during its growth phase. To demonstrate the biotechnological benefit of E. coli harboring a high intracellular ATP concentration, we compared the recombinant protein synthesis of a soluble protein (enhanced green fluorescence protein, GFP) with that of a secretory protein (alkaline protease, AP), under control of the T7 promoter in E. coli BL21(DE3) overexpressing PCK. According to the batch fermentations, the strain overexpressing PCK produced more GFP and AP with a lower increase in biomass than the control strain. In a chemostat culture (D = 0.7 h−1), the GFP production in the PCK overexpressing strain was 99.0 ± 4.31 mg/g cell, with a biomass of 0.22 g/L, while that of the control strain was 53.5 ± 3.07 mg/g cell, with a biomass of 0.35 g/L. These results indicate that the PCK overexpressing E. coli strain harboring high intracellular levels of ATP can be useful as a protein-synthesizing host. The potential uses of the strain and associated rationale are discussed.
Keywords: High intracellular ATP concentration; Recombinant protein expression; Cellular energy
Enhanced electrode-reducing rate during the enrichment process in an air-cathode microbial fuel cell by Shun’ichi Ishii; Bruce E. Logan; Yuji Sekiguchi (pp. 1087-1094).
The improvement in electricity generation during the enrichment process of a microbial consortium was analyzed using an air-cathode microbial fuel cell (MFC) repeatedly fed with acetate that was originally inoculated with sludge from an anaerobic digester. The anodic maximum current density produced by the anode biofilm increased from 0.12 mA/cm2 at day 28 to 1.12 mA/cm2 at day 105. However, the microbial cell density on the carbon cloth anode increased only three times throughout this same time period from 0.21 to 0.69 mg protein/cm2, indicating that the biocatalytic activity of the consortium was also enhanced. The microbial activity was calculated to have a per biomass anode-reducing rate of 374 μmol electron g protein−1 min−1 at day 28 and 1,002 μmol electron g protein−1 min−1 at day 105. A bacterial community analysis of the anode biofilm revealed that the dominant phylotype, which was closely related to the known exoelectrogenic bacterium, Geobacter sulfurreducens, showed an increase in abundance from 32% to 70% of the total microbial cells. Fluorescent in situ hybridization observation also showed the increase of Geobacter-like phylotypes from 53% to 72%. These results suggest that the improvement of microbial current generation in microbial fuel cells is a function of both microbial cell growth on the electrode and changes in the bacterial community highly dominated by a known exoelectrogenic bacterium during the enrichment process.
Keywords: Microbial fuel cell; Current density; Biofilm; Electrode-reducing rate
Enhanced electrode-reducing rate during the enrichment process in an air-cathode microbial fuel cell by Shun’ichi Ishii; Bruce E. Logan; Yuji Sekiguchi (pp. 1087-1094).
The improvement in electricity generation during the enrichment process of a microbial consortium was analyzed using an air-cathode microbial fuel cell (MFC) repeatedly fed with acetate that was originally inoculated with sludge from an anaerobic digester. The anodic maximum current density produced by the anode biofilm increased from 0.12 mA/cm2 at day 28 to 1.12 mA/cm2 at day 105. However, the microbial cell density on the carbon cloth anode increased only three times throughout this same time period from 0.21 to 0.69 mg protein/cm2, indicating that the biocatalytic activity of the consortium was also enhanced. The microbial activity was calculated to have a per biomass anode-reducing rate of 374 μmol electron g protein−1 min−1 at day 28 and 1,002 μmol electron g protein−1 min−1 at day 105. A bacterial community analysis of the anode biofilm revealed that the dominant phylotype, which was closely related to the known exoelectrogenic bacterium, Geobacter sulfurreducens, showed an increase in abundance from 32% to 70% of the total microbial cells. Fluorescent in situ hybridization observation also showed the increase of Geobacter-like phylotypes from 53% to 72%. These results suggest that the improvement of microbial current generation in microbial fuel cells is a function of both microbial cell growth on the electrode and changes in the bacterial community highly dominated by a known exoelectrogenic bacterium during the enrichment process.
Keywords: Microbial fuel cell; Current density; Biofilm; Electrode-reducing rate
Characterization of the mitochondrial NAD+-dependent isocitrate dehydrogenase of the oleaginous yeast Rhodosporidium toruloides by Fan Yang; Sufang Zhang; Yongjin J. Zhou; Zhiwei Zhu; Xinping Lin; Zongbao K. Zhao (pp. 1095-1105).
Early biochemical studies have demonstrated that lipid accumulation by oleaginous yeasts is linked to the activity of the NAD+-dependent isocitrate dehydrogenase (Idh). However, molecular study of Idh of oleaginous microorganisms remains limited. Here, we present the cloning of a mitochondrial NAD+-specific Idh from Rhodosporidium toruloides (RtIdh), an excellent microbial lipid producer that uses carbohydrates as the carbon source. The evolutionary relationship analyses among RtIdhs and other yeast Idhs revealed that RtIdh had a closer relationship with the Idhs of Ustilago maydis and Schizophyllum commune. We expressed the RtIDH gene in the yeast Saccharomyces cerevisiae idhΔ mutant. Under the nitrogen-limited condition, the intracellular lipid content and extracellular citrate concentration of the culture of the S. cerevisiae idhΔ carrying the RtIDH gene increased as the carbon/nitrogen molar ratio of the media increased, while the wild-type S. cerevisiae strain showed no correlation. Our data provided valuable information for elucidating the molecular mechanism of microbial oleaginicity and for engineering microorganisms to produce metabolites of fatty acid pathway.
Keywords: Rhodosporidium toruloides ; Microbial lipid; NAD+-specific isocitrate dehydrogenase; Nitrogen limitation; Oleaginous yeast
Characterization of the mitochondrial NAD+-dependent isocitrate dehydrogenase of the oleaginous yeast Rhodosporidium toruloides by Fan Yang; Sufang Zhang; Yongjin J. Zhou; Zhiwei Zhu; Xinping Lin; Zongbao K. Zhao (pp. 1095-1105).
Early biochemical studies have demonstrated that lipid accumulation by oleaginous yeasts is linked to the activity of the NAD+-dependent isocitrate dehydrogenase (Idh). However, molecular study of Idh of oleaginous microorganisms remains limited. Here, we present the cloning of a mitochondrial NAD+-specific Idh from Rhodosporidium toruloides (RtIdh), an excellent microbial lipid producer that uses carbohydrates as the carbon source. The evolutionary relationship analyses among RtIdhs and other yeast Idhs revealed that RtIdh had a closer relationship with the Idhs of Ustilago maydis and Schizophyllum commune. We expressed the RtIDH gene in the yeast Saccharomyces cerevisiae idhΔ mutant. Under the nitrogen-limited condition, the intracellular lipid content and extracellular citrate concentration of the culture of the S. cerevisiae idhΔ carrying the RtIDH gene increased as the carbon/nitrogen molar ratio of the media increased, while the wild-type S. cerevisiae strain showed no correlation. Our data provided valuable information for elucidating the molecular mechanism of microbial oleaginicity and for engineering microorganisms to produce metabolites of fatty acid pathway.
Keywords: Rhodosporidium toruloides ; Microbial lipid; NAD+-specific isocitrate dehydrogenase; Nitrogen limitation; Oleaginous yeast
Engineering global transcription factor cyclic AMP receptor protein of Escherichia coli for improved 1-butanol tolerance by Hongfang Zhang; Huiqing Chong; Chi Bun Ching; Hao Song; Rongrong Jiang (pp. 1107-1117).
One major challenge in biofuel production, including biobutanol production, is the low tolerance of the microbial host towards increasing biofuel concentration during fermentation. Here, we have demonstrated that Escherichia coli 1-butanol tolerance can be greatly enhanced through random mutagenesis of global transcription factor cyclic AMP receptor protein (CRP). Four mutants (MT1–MT4) with elevated 1-butanol tolerance were isolated from error-prone PCR libraries through an enrichment screening. A DNA shuffling library was then constructed using MT1–MT4 as templates and one mutant (MT5) that exhibited the best tolerance ability among all variants was selected. In the presence of 0.8 % (v/v, 6.5 g/l) 1-butanol, the growth rate of MT5 was found to be 0.28 h−1 while that of wild type was 0.20 h−1. When 1-butanol concentration increased to 1.2 % (9.7 g/l), the growth rate of MT5 (0.18 h−1) became twice that of the wild type (0.09 h−1). Microbial adhesion to hydrocarbon test showed that cell surface of MT5 was less hydrophobic and its cell length became significantly longer in the presence of 1-butanol, as observed by scanning electron microscopy. Quantitative real-time reverse transcription PCR analysis revealed that several CRP regulated, 1-butanol stress response related genes (rpoH, ompF, sodA, manX, male, and marA) demonstrated differential expression in MT5 in the presence or absence of 1-butanol. In conclusion, direct manipulation of the transcript profile through engineering global transcription factor CRP can provide a useful tool in strain engineering.
Keywords: 1-butanol tolerance; cAMP receptor protein; Error-prone PCR; DNA shuffling; Transcriptional engineering; Global transcription factor
Engineering global transcription factor cyclic AMP receptor protein of Escherichia coli for improved 1-butanol tolerance by Hongfang Zhang; Huiqing Chong; Chi Bun Ching; Hao Song; Rongrong Jiang (pp. 1107-1117).
One major challenge in biofuel production, including biobutanol production, is the low tolerance of the microbial host towards increasing biofuel concentration during fermentation. Here, we have demonstrated that Escherichia coli 1-butanol tolerance can be greatly enhanced through random mutagenesis of global transcription factor cyclic AMP receptor protein (CRP). Four mutants (MT1–MT4) with elevated 1-butanol tolerance were isolated from error-prone PCR libraries through an enrichment screening. A DNA shuffling library was then constructed using MT1–MT4 as templates and one mutant (MT5) that exhibited the best tolerance ability among all variants was selected. In the presence of 0.8 % (v/v, 6.5 g/l) 1-butanol, the growth rate of MT5 was found to be 0.28 h−1 while that of wild type was 0.20 h−1. When 1-butanol concentration increased to 1.2 % (9.7 g/l), the growth rate of MT5 (0.18 h−1) became twice that of the wild type (0.09 h−1). Microbial adhesion to hydrocarbon test showed that cell surface of MT5 was less hydrophobic and its cell length became significantly longer in the presence of 1-butanol, as observed by scanning electron microscopy. Quantitative real-time reverse transcription PCR analysis revealed that several CRP regulated, 1-butanol stress response related genes (rpoH, ompF, sodA, manX, male, and marA) demonstrated differential expression in MT5 in the presence or absence of 1-butanol. In conclusion, direct manipulation of the transcript profile through engineering global transcription factor CRP can provide a useful tool in strain engineering.
Keywords: 1-butanol tolerance; cAMP receptor protein; Error-prone PCR; DNA shuffling; Transcriptional engineering; Global transcription factor