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Jay D Keasling - One of the best experts on this subject based on the ideXlab platform.
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Autonomous control of metabolic state by a quorum sensing (QS)-mediated regulator for Bisabolene production in engineered E. coli.
Metabolic engineering, 2017Co-Authors: Eun-mi Kim, Jay D Keasling, Han Min Woo, Tian Tian, Suzan Yilmaz, Pouya Javidpour, Taek Soon LeeAbstract:Inducible gene expression systems are widely used in microbial host strains for protein and commodity chemical production because of their extensive characterization and ease of use. However, some of these systems have disadvantages such as leaky expression, lack of dynamic control, and the prohibitively high costs of inducers associated with large-scale production. Quorum sensing (QS) systems in bacteria control gene expression in response to population density, and the LuxI/R system from Vibrio fischeri is a well-studied example. A QS system could be ideal for biofuel production strains as it is self-regulated and does not require the addition of inducer compounds, which reduce operational costs for inducer. In this study, a QS system was developed for inducer-free production of the biofuel compound Bisabolene from engineered E. coli. Seven variants of the Sensor plasmid, which carry the luxI-luxR genes, and four variants of the Response plasmid, which carry Bisabolene producing pathway genes under the control of the PluxI promoter, were designed for optimization of Bisabolene production. Furthermore, a chromosome-integrated QS strain was engineered with the best combination of Sensor and Response plasmid and produced Bisabolene at a titer of 1.1g/L without addition of external inducers. This is a 44% improvement from our previous inducible system. The QS strain also displayed higher homogeneity in gene expression and isoprenoid production compared to an inducible-system strain.
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Use of Nonionic Surfactants for Improvement of Terpene Production in Saccharomyces cerevisiae
Applied and environmental microbiology, 2014Co-Authors: James Kirby, Rossana Chan, Minobu Nishimoto, Ruthie W. N. Chow, Venkata N. Pasumarthi, Leanne Jade G. Chan, Christopher J. Petzold, Jay D KeaslingAbstract:To facilitate enzyme and pathway engineering, a selection was developed for improved sesquiterpene titers in Saccharomyces cerevisiae. α-Bisabolene, a candidate advanced biofuel, was found to protect yeast against the disruptive action of nonionic surfactants such as Tween 20 (T20). An experiment employing competition between two strains of yeast, one of which makes twice as much Bisabolene as the other, demonstrated that growth in the presence of T20 provided sufficient selective pressure to enrich the high-titer strain to form 97% of the population. Following this, various methods were used to mutagenize the Bisabolene synthase (BIS) coding sequence, coupled with selection by subculturing in the presence of T20. Mutagenesis targeting the BIS active site did not yield an improvement in Bisabolene titers, although mutants were found which made a mixture of α-Bisabolene and β-farnesene, another candidate biofuel. Based on evidence that the 3' end of the BIS mRNA may be unstable in yeast, we randomly recoded the last 20 amino acids of the enzyme and, following selection in T20, found a variant which increased specific production of Bisabolene by more than 30%. Since T20 could enrich a mixed population, efficiently removing strains that produced little or no Bisabolene, we investigated whether it could also be applied to sustain high product titers in a monoculture for an extended period. Cultures grown in the presence of T20 for 14 days produced Bisabolene at titers up to 4-fold higher than cultures grown with an overlay of dodecane, used to sequester the terpene product, and 20-fold higher than cultures grown without dodecane.
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carotenoid based phenotypic screen of the yeast deletion collection reveals new genes with roles in isoprenoid production
Metabolic Engineering, 2013Co-Authors: Bilge Ozaydin, Helcio Burd, Jay D KeaslingAbstract:Abstract Beside their essential cellular functions, isoprenoids have value as pharmaceuticals, nutriceuticals, pesticides, and fuel alternatives. Engineering microorganisms for production of isoprenoids is relatively easy, sustainable, and cost effective in comparison to chemical synthesis or extraction from natural producers. We introduced genes encoding carotenoid biosynthetic enzymes into the haploid yeast deletion collection to identify gene deletions that improved isoprenoid production. Deletions that showed significant improvement in carotenoid production were further screened for production of Bisabolene, an isoprenoid alternative to petroleum-derived diesel. Combining those deletions with other mevalonate pathway modifications increased production of Bisabolene from 40 mg/L to 800 mg/L in shake-flask cultures. In a fermentation process, this engineered strain produced 5.2 g/L of Bisabolene.
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carotenoid based phenotypic screen of the yeast deletion collection reveals new genes with roles in isoprenoid production
Metabolic Engineering, 2013Co-Authors: Bilge Ozaydin, Helcio Burd, Jay D KeaslingAbstract:Abstract Beside their essential cellular functions, isoprenoids have value as pharmaceuticals, nutriceuticals, pesticides, and fuel alternatives. Engineering microorganisms for production of isoprenoids is relatively easy, sustainable, and cost effective in comparison to chemical synthesis or extraction from natural producers. We introduced genes encoding carotenoid biosynthetic enzymes into the haploid yeast deletion collection to identify gene deletions that improved isoprenoid production. Deletions that showed significant improvement in carotenoid production were further screened for production of Bisabolene, an isoprenoid alternative to petroleum-derived diesel. Combining those deletions with other mevalonate pathway modifications increased production of Bisabolene from 40 mg/L to 800 mg/L in shake-flask cultures. In a fermentation process, this engineered strain produced 5.2 g/L of Bisabolene.
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Structure of a three-domain sesquiterpene synthase: a prospective target for advanced biofuels production.
Structure (London England : 1993), 2011Co-Authors: Ryan P. Mcandrew, Jay D Keasling, Pamela Peralta-yahya, Andy Degiovanni, Jose Henrique Pereira, Masood Z. Hadi, Paul D. AdamsAbstract:The sesquiterpene Bisabolene was recently identified as a biosynthetic precursor to bisabolane, an advanced biofuel with physicochemical properties similar to those of D2 diesel. High-titer microbial Bisabolene production was achieved using Abies grandis α-Bisabolene synthase (AgBIS). Here, we report the structure of AgBIS, a three-domain plant sesquiterpene synthase, crystallized in its apo form and bound to five different inhibitors. Structural and biochemical characterization of the AgBIS terpene synthase Class I active site leads us to propose a catalytic mechanism for the cyclization of farnesyl diphosphate into Bisabolene via a bisabolyl cation intermediate. Further, we describe the nonfunctional AgBIS Class II active site whose high similarity to bifunctional diterpene synthases makes it an important link in understanding terpene synthase evolution. Practically, the AgBIS crystal structure is important in future protein engineering efforts to increase the microbial production of Bisabolene.
Rossana Chan - One of the best experts on this subject based on the ideXlab platform.
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Use of Nonionic Surfactants for Improvement of Terpene Production in Saccharomyces cerevisiae
Applied and environmental microbiology, 2014Co-Authors: James Kirby, Rossana Chan, Minobu Nishimoto, Ruthie W. N. Chow, Venkata N. Pasumarthi, Leanne Jade G. Chan, Christopher J. Petzold, Jay D KeaslingAbstract:To facilitate enzyme and pathway engineering, a selection was developed for improved sesquiterpene titers in Saccharomyces cerevisiae. α-Bisabolene, a candidate advanced biofuel, was found to protect yeast against the disruptive action of nonionic surfactants such as Tween 20 (T20). An experiment employing competition between two strains of yeast, one of which makes twice as much Bisabolene as the other, demonstrated that growth in the presence of T20 provided sufficient selective pressure to enrich the high-titer strain to form 97% of the population. Following this, various methods were used to mutagenize the Bisabolene synthase (BIS) coding sequence, coupled with selection by subculturing in the presence of T20. Mutagenesis targeting the BIS active site did not yield an improvement in Bisabolene titers, although mutants were found which made a mixture of α-Bisabolene and β-farnesene, another candidate biofuel. Based on evidence that the 3' end of the BIS mRNA may be unstable in yeast, we randomly recoded the last 20 amino acids of the enzyme and, following selection in T20, found a variant which increased specific production of Bisabolene by more than 30%. Since T20 could enrich a mixed population, efficiently removing strains that produced little or no Bisabolene, we investigated whether it could also be applied to sustain high product titers in a monoculture for an extended period. Cultures grown in the presence of T20 for 14 days produced Bisabolene at titers up to 4-fold higher than cultures grown with an overlay of dodecane, used to sequester the terpene product, and 20-fold higher than cultures grown without dodecane.
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Identification and microbial production of a terpene-based advanced biofuel
Nature Communications, 2011Co-Authors: Pamela P. Peralta-yahya, Mario Ouellet, Rossana Chan, Aindrila Mukhopadhyay, Jay D KeaslingAbstract:Rising petroleum costs, trade imbalances and environmental concerns have stimulated efforts to advance the microbial production of fuels from lignocellulosic biomass. Here we identify a novel biosynthetic alternative to D2 diesel fuel, bisabolane, and engineer microbial platforms for the production of its immediate precursor, Bisabolene. First, we identify bisabolane as an alternative to D2 diesel by measuring the fuel properties of chemically hydrogenated commercial Bisabolene. Then, via a combination of enzyme screening and metabolic engineering, we obtain a more than tenfold increase in Bisabolene titers in Escherichia coli to >900 mg l^−1. We produce Bisabolene in Saccharomyces cerevisiae (>900 mg l^−1), a widely used platform for the production of ethanol. Finally, we chemically hydrogenate biosynthetic Bisabolene into bisabolane. This work presents a framework for the identification of novel terpene-based advanced biofuels and the rapid engineering of microbial farnesyl diphosphate-overproducing platforms for the production of biofuels. Advanced biofuels with comparable properties to petroleum-based fuels could be microbially produced from lignocellulosic biomass. In this study, Escherichia coli is engineered to produce Bisabolene, the immediate precursor of bisabolane, a biosynthetic alternative to D2 diesel.
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identification and microbial production of a terpene based advanced biofuel
Nature Communications, 2011Co-Authors: Pamela Peraltayahya, Jay D Keasling, Mario Ouellet, Rossana Chan, Aindrila MukhopadhyayAbstract:Rising petroleum costs, trade imbalances and environmental concerns have stimulated efforts to advance the microbial production of fuels from lignocellulosic biomass. Here we identify a novel biosynthetic alternative to D2 diesel fuel, bisabolane, and engineer microbial platforms for the production of its immediate precursor, Bisabolene. First, we identify bisabolane as an alternative to D2 diesel by measuring the fuel properties of chemically hydrogenated commercial Bisabolene. Then, via a combination of enzyme screening and metabolic engineering, we obtain a more than tenfold increase in Bisabolene titers in Escherichia coli to >900 mg l(-1). We produce Bisabolene in Saccharomyces cerevisiae (>900 mg l(-1)), a widely used platform for the production of ethanol. Finally, we chemically hydrogenate biosynthetic Bisabolene into bisabolane. This work presents a framework for the identification of novel terpene-based advanced biofuels and the rapid engineering of microbial farnesyl diphosphate-overproducing platforms for the production of biofuels.
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identification and microbial production of a terpene based advanced biofuel
Nature Communications, 2011Co-Authors: Jay D Keasling, Mario Ouellet, Rossana Chan, Aindrila Mukhopadhyay, Pamela PeraltayahyaAbstract:Advanced biofuels with comparable properties to petroleum-based fuels could be microbially produced from lignocellulosic biomass. In this study, Escherichia coli is engineered to produce Bisabolene, the immediate precursor of bisabolane, a biosynthetic alternative to D2 diesel.
James S. Chickos - One of the best experts on this subject based on the ideXlab platform.
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the vapor pressure and vaporization enthalpy of β elemene and β Bisabolene by correlation gas chromatography
The Journal of Chemical Thermodynamics, 2020Co-Authors: Dustin Barton, James S. ChickosAbstract:Abstract The vaporization enthalpies and vapor pressures of (−) β-elemene and (−) β-Bisabolene are evaluated by correlation gas chromatography using two sets of standards, a mixture of various cyclic hydrocarbons and a series of n-alkanes. Both compounds are sesquiterpenes found in a variety of plants. β-Elemene has been used in China for the treatment of a variety of cancers. β-Bisabolene is used as a flavoring agent and it has also been shown to exhibit cytotoxicity in breast cancer cell lines, both in vitro and in vivo. Vaporization enthalpies of [(66.8 ± 3.0) and (73.9 ± 3.0)] kJ·mol−1 and vapor pressures of [(3.1 ± 1.0) and (0.72 ± 0.2)] Pa at T = 298.15 K have been evaluated for (−) β-elemene and (−) β-Bisabolene, respectively. Vapor pressures were also evaluated as a function of temperature from T = (298.15–450) K and fit to a second order polynomial.
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The vapor pressure and vaporization enthalpy of (−) β-Elemene and (−) β-Bisabolene by correlation gas chromatography
The Journal of Chemical Thermodynamics, 2020Co-Authors: Dustin Barton, James S. ChickosAbstract:Abstract The vaporization enthalpies and vapor pressures of (−) β-elemene and (−) β-Bisabolene are evaluated by correlation gas chromatography using two sets of standards, a mixture of various cyclic hydrocarbons and a series of n-alkanes. Both compounds are sesquiterpenes found in a variety of plants. β-Elemene has been used in China for the treatment of a variety of cancers. β-Bisabolene is used as a flavoring agent and it has also been shown to exhibit cytotoxicity in breast cancer cell lines, both in vitro and in vivo. Vaporization enthalpies of [(66.8 ± 3.0) and (73.9 ± 3.0)] kJ·mol−1 and vapor pressures of [(3.1 ± 1.0) and (0.72 ± 0.2)] Pa at T = 298.15 K have been evaluated for (−) β-elemene and (−) β-Bisabolene, respectively. Vapor pressures were also evaluated as a function of temperature from T = (298.15–450) K and fit to a second order polynomial.
Aindrila Mukhopadhyay - One of the best experts on this subject based on the ideXlab platform.
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Identification and microbial production of a terpene-based advanced biofuel
Nature Communications, 2011Co-Authors: Pamela P. Peralta-yahya, Mario Ouellet, Rossana Chan, Aindrila Mukhopadhyay, Jay D KeaslingAbstract:Rising petroleum costs, trade imbalances and environmental concerns have stimulated efforts to advance the microbial production of fuels from lignocellulosic biomass. Here we identify a novel biosynthetic alternative to D2 diesel fuel, bisabolane, and engineer microbial platforms for the production of its immediate precursor, Bisabolene. First, we identify bisabolane as an alternative to D2 diesel by measuring the fuel properties of chemically hydrogenated commercial Bisabolene. Then, via a combination of enzyme screening and metabolic engineering, we obtain a more than tenfold increase in Bisabolene titers in Escherichia coli to >900 mg l^−1. We produce Bisabolene in Saccharomyces cerevisiae (>900 mg l^−1), a widely used platform for the production of ethanol. Finally, we chemically hydrogenate biosynthetic Bisabolene into bisabolane. This work presents a framework for the identification of novel terpene-based advanced biofuels and the rapid engineering of microbial farnesyl diphosphate-overproducing platforms for the production of biofuels. Advanced biofuels with comparable properties to petroleum-based fuels could be microbially produced from lignocellulosic biomass. In this study, Escherichia coli is engineered to produce Bisabolene, the immediate precursor of bisabolane, a biosynthetic alternative to D2 diesel.
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identification and microbial production of a terpene based advanced biofuel
Nature Communications, 2011Co-Authors: Pamela Peraltayahya, Jay D Keasling, Mario Ouellet, Rossana Chan, Aindrila MukhopadhyayAbstract:Rising petroleum costs, trade imbalances and environmental concerns have stimulated efforts to advance the microbial production of fuels from lignocellulosic biomass. Here we identify a novel biosynthetic alternative to D2 diesel fuel, bisabolane, and engineer microbial platforms for the production of its immediate precursor, Bisabolene. First, we identify bisabolane as an alternative to D2 diesel by measuring the fuel properties of chemically hydrogenated commercial Bisabolene. Then, via a combination of enzyme screening and metabolic engineering, we obtain a more than tenfold increase in Bisabolene titers in Escherichia coli to >900 mg l(-1). We produce Bisabolene in Saccharomyces cerevisiae (>900 mg l(-1)), a widely used platform for the production of ethanol. Finally, we chemically hydrogenate biosynthetic Bisabolene into bisabolane. This work presents a framework for the identification of novel terpene-based advanced biofuels and the rapid engineering of microbial farnesyl diphosphate-overproducing platforms for the production of biofuels.
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identification and microbial production of a terpene based advanced biofuel
Nature Communications, 2011Co-Authors: Jay D Keasling, Mario Ouellet, Rossana Chan, Aindrila Mukhopadhyay, Pamela PeraltayahyaAbstract:Advanced biofuels with comparable properties to petroleum-based fuels could be microbially produced from lignocellulosic biomass. In this study, Escherichia coli is engineered to produce Bisabolene, the immediate precursor of bisabolane, a biosynthetic alternative to D2 diesel.
Mario Ouellet - One of the best experts on this subject based on the ideXlab platform.
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Identification and microbial production of a terpene-based advanced biofuel
Nature Communications, 2011Co-Authors: Pamela P. Peralta-yahya, Mario Ouellet, Rossana Chan, Aindrila Mukhopadhyay, Jay D KeaslingAbstract:Rising petroleum costs, trade imbalances and environmental concerns have stimulated efforts to advance the microbial production of fuels from lignocellulosic biomass. Here we identify a novel biosynthetic alternative to D2 diesel fuel, bisabolane, and engineer microbial platforms for the production of its immediate precursor, Bisabolene. First, we identify bisabolane as an alternative to D2 diesel by measuring the fuel properties of chemically hydrogenated commercial Bisabolene. Then, via a combination of enzyme screening and metabolic engineering, we obtain a more than tenfold increase in Bisabolene titers in Escherichia coli to >900 mg l^−1. We produce Bisabolene in Saccharomyces cerevisiae (>900 mg l^−1), a widely used platform for the production of ethanol. Finally, we chemically hydrogenate biosynthetic Bisabolene into bisabolane. This work presents a framework for the identification of novel terpene-based advanced biofuels and the rapid engineering of microbial farnesyl diphosphate-overproducing platforms for the production of biofuels. Advanced biofuels with comparable properties to petroleum-based fuels could be microbially produced from lignocellulosic biomass. In this study, Escherichia coli is engineered to produce Bisabolene, the immediate precursor of bisabolane, a biosynthetic alternative to D2 diesel.
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identification and microbial production of a terpene based advanced biofuel
Nature Communications, 2011Co-Authors: Pamela Peraltayahya, Jay D Keasling, Mario Ouellet, Rossana Chan, Aindrila MukhopadhyayAbstract:Rising petroleum costs, trade imbalances and environmental concerns have stimulated efforts to advance the microbial production of fuels from lignocellulosic biomass. Here we identify a novel biosynthetic alternative to D2 diesel fuel, bisabolane, and engineer microbial platforms for the production of its immediate precursor, Bisabolene. First, we identify bisabolane as an alternative to D2 diesel by measuring the fuel properties of chemically hydrogenated commercial Bisabolene. Then, via a combination of enzyme screening and metabolic engineering, we obtain a more than tenfold increase in Bisabolene titers in Escherichia coli to >900 mg l(-1). We produce Bisabolene in Saccharomyces cerevisiae (>900 mg l(-1)), a widely used platform for the production of ethanol. Finally, we chemically hydrogenate biosynthetic Bisabolene into bisabolane. This work presents a framework for the identification of novel terpene-based advanced biofuels and the rapid engineering of microbial farnesyl diphosphate-overproducing platforms for the production of biofuels.
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identification and microbial production of a terpene based advanced biofuel
Nature Communications, 2011Co-Authors: Jay D Keasling, Mario Ouellet, Rossana Chan, Aindrila Mukhopadhyay, Pamela PeraltayahyaAbstract:Advanced biofuels with comparable properties to petroleum-based fuels could be microbially produced from lignocellulosic biomass. In this study, Escherichia coli is engineered to produce Bisabolene, the immediate precursor of bisabolane, a biosynthetic alternative to D2 diesel.