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Perspective article on the future of biofuels using microbial platforms in the January 2107 edition of the Biofuels International Magazine. http://www.jbei.org/wp-content/uploads/2017/02/34-35_biofuels_Jan-Feb_2017-1-1.pdf

Perspective article on the future of biofuels using microbial platforms in the January 2107 edition of the Biofuels International Magazine. http://www.jbei.org/wp-content/uploads/2017/02/34-35_biofuels_Jan-Feb_2017-1-1.pdf

The scope of the project is to investigate efflux pump systems in engineered host microorganisms, such as E. coli, and develop a pump engineered to export a target compound. To initiate the project in coordination with other TOTAL driven projects, the first target compound to be studied was 1-hexene. However, we were investigating other chemicals as Styrene. The main goal of the project was to generate a set of optimized efflux pump systems for microorganisms (E. coli and Streptomyces or other host) engineered to contain biosynthetic pathways to export large titers of target compounds that are toxic (or accumulate and push back biosynthesis) to the host cell. An optimized microbial host will utilize specific and efficient cell wall located pumps to extrude harmful target compounds and enable greater production of these compounds.

The scope of the project is to investigate efflux pump systems in engineered host microorganisms, such as E. coli, and develop a pump engineered to export a target compound. To initiate the project in coordination with other TOTAL driven projects, the first target compound to be studied was 1-hexene. However, we were investigating other chemicals as Styrene. The main goal of the project was to generate a set of optimized efflux pump systems for microorganisms (E. coli and Streptomyces or other host) engineered to contain biosynthetic pathways to export large titers of target compounds that are toxic (or accumulate and push back biosynthesis) to the host cell. An optimized microbial host will utilize specific and efficient cell wall located pumps to extrude harmful target compounds and enable greater production of these compounds.

During microbial production of solvent-like compounds, such as advanced biofuels and bulk chemicals, accumulation of the final product can negatively impact the cultivation of the host microbe and limit the production levels. Consequently, improving solvent tolerance is becoming an essential aspect of engineering microbial production strains. Mechanisms ranging from chaperones to transcriptional factors have been used to obtain solvent-tolerant strains. However, alleviating growth inhibition does not invariably result in increased production. Transporters specifically have emerged as a powerful category of proteins that bestow tolerance and often improve production but are difficult targets for cellular expression. Here we review strain engineering, primarily as it pertains to bacterial solvent tolerance, and the benefits and challenges associated with the expression of membrane-localized transporters in improving solvent tolerance and production.

During microbial production of solvent-like compounds, such as advanced biofuels and bulk chemicals, accumulation of the final product can negatively impact the cultivation of the host microbe and limit the production levels. Consequently, improving solvent tolerance is becoming an essential aspect of engineering microbial production strains. Mechanisms ranging from chaperones to transcriptional factors have been used to obtain solvent-tolerant strains. However, alleviating growth inhibition does not invariably result in increased production. Transporters specifically have emerged as a powerful category of proteins that bestow tolerance and often improve production but are difficult targets for cellular expression. Here we review strain engineering, primarily as it pertains to bacterial solvent tolerance, and the benefits and challenges associated with the expression of membrane-localized transporters in improving solvent tolerance and production.

Microorganisms have been rich sources for natural products, some of which have found use as fuels, commodity chemicals, specialty chemicals, polymers, and drugs, to name a few. The recent interest in production of transportation fuels from renewable resources has catalyzed numerous research endeavors that focus on developing microbial systems for production of such natural products. Eliminating bottlenecks in microbial metabolic pathways and alleviating the stresses due to production of these chemicals are crucial in the generation of robust and efficient production hosts. The use of systems-level studies makes it possible to comprehensively understand the impact of pathway engineering within the context of the entire host metabolism, to diagnose stresses due to product synthesis, and provides the rationale to cost-effectively engineer optimal industrial microorganisms.

Hypothetical and conserved hypothetical genes account for >30percent of sequenced bacterial genomes. For the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, 348 of the 3534 genes are annotated as conserved hypothetical (9.7percent) with 889 hypothetical genes (25.0percent). Given this large genome fraction, it is plausible that some genes serve critical cellular functions. The goals of this study were to determine which genes can be expressed and to provide a more functionally based annotation. To accomplish this, expression profiles of the 1237 hypothetical and conserved hypothetical genes were obtained from transcriptomic datasets of 10 environmental stresses, complimented with iTRAQ proteomic data. Genes were divided into putatively polycistronic operons and those predicted to be monocistronic, then classified by basal expression levels and grouped according to changes in expression for one or multiple stresses. 1219 of these genes were transcribed with 265 proteins detected. There was no evidence for expression of 17 predicted genes. Except for the latter, annotation of all monocistronic genes was expanded using the above criteria and COG information. Polycistronic genes were annotated with expression information including proximity to more confidently annotated genes. Two targeted deletion mutants were used as test cases to determine the accuracy of the inferred functional annotations.

Hypothetical and conserved hypothetical genes account for >30percent of sequenced bacterial genomes. For the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, 348 of the 3534 genes are annotated as conserved hypothetical (9.7percent) with 889 hypothetical genes (25.0percent). Given this large genome fraction, it is plausible that some genes serve critical cellular functions. The goals of this study were to determine which genes can be expressed and to provide a more functionally based annotation. To accomplish this, expression profiles of the 1237 hypothetical and conserved hypothetical genes were obtained from transcriptomic datasets of 10 environmental stresses, complimented with iTRAQ proteomic data. Genes were divided into putatively polycistronic operons and those predicted to be monocistronic, then classified by basal expression levels and grouped according to changes in expression for one or multiple stresses. 1219 of these genes were transcribed with 265 proteins detected. There was no evidence for expression of 17 predicted genes. Except for the latter, annotation of all monocistronic genes was expanded using the above criteria and COG information. Polycistronic genes were annotated with expression information including proximity to more confidently annotated genes. Two targeted deletion mutants were used as test cases to determine the accuracy of the inferred functional annotations.

Microorganisms have been rich sources for natural products, some of which have found use as fuels, commodity chemicals, specialty chemicals, polymers, and drugs, to name a few. The recent interest in production of transportation fuels from renewable resources has catalyzed numerous research endeavors that focus on developing microbial systems for production of such natural products. Eliminating bottlenecks in microbial metabolic pathways and alleviating the stresses due to production of these chemicals are crucial in the generation of robust and efficient production hosts. The use of systems-level studies makes it possible to comprehensively understand the impact of pathway engineering within the context of the entire host metabolism, to diagnose stresses due to product synthesis, and provides the rationale to cost-effectively engineer optimal industrial microorganisms.

The overview of changes in protein levels or states in response to a growth condition, stress, mutation or metabolic engineering is invaluable in understanding the physiology of a microbial system. The lipid profile of the cell is similarly a valuable diagnostic of the cellular response and health, especially in context of survival in a fluctuating environment. To obtain comprehensive cellular models, post-transcriptional cell wide surveys at the levels of proteins and lipids are required. Both these fields have been greatly bolstered by the development of high throughput methods using mass spectrometry. Multiple strategies now exist for the identification of proteins, and numerous workflows to quantify protein abundance have also been developed. Cellular profiling such as these allows us to assess the potential of a microbial system for environmental applications such as bioremediation and bio-energy.

A fundamental goal of the Environmental Stress Pathway Project (ESPP) of the Virtual Institute of Stress and survival (VIMSS) is a rigorous understanding of Desulfovibrio vulgaris Hildenborough physiology and its ability to survive in its environment. Such knowledge will be critical in discerning the biogeochemistry at metal contaminated sites, for bioremediation and natural attenuation for toxic metals. The Functional Genomics and Imaging Core (FGIC) focuses on the measurement of these responses at a cell wide level using systems biology approaches. Progress in the last one year built upon our optimized pipeline for generating biomass for various functional genomics studies and utilized improved genetic methods. Numerous additional transcriptomics data sets were added to our compendium of stress response studies. These included peroxide stress, low oxygen stress, high and low pH stress and alteration of growth conditions (e.g presence of methionine, alternate electron donors etc). To understand how genotype and environment interact to determine the phenotype and fitness of an organism, a long-term evolution experiment was also conducted to examine the dynamics and adaptation of D. vulgaris under extended salt exposure. For many of these stresses iTRAQ based quantitative proteomics and CE-MS based metabolite studies were also conducted. Improved genetic methods were employed to create several critical knock out mutants (e.g. echA, qmoABC and tatA) and several were characterized via growth and transcriptomics studies. Progress was also made in extending transcriptomics analysis to examine alternate D. vulgaris physiological states such as in biofilms and growth in syntrophic co-culture with Methanococcus maripaludis. Methods to conduct iTRAQ proteomics and stable isotopomer (13C) based metabolic flux analysis were also developed for studying co-cultures. A novel FTICR-MS based method for a comparative 12C/13C based metabolite analysis is being developed and will enab

A fundamental goal of the Environmental Stress Pathway Project (ESPP) of the Virtual Institute of Stress and survival (VIMSS) is a rigorous understanding of Desulfovibrio vulgaris Hildenborough physiology and its ability to survive in its environment. Such knowledge will be critical in discerning the biogeochemistry at metal contaminated sites, for bioremediation and natural attenuation for toxic metals. The Functional Genomics and Imaging Core (FGIC) focuses on the measurement of these responses at a cell wide level using systems biology approaches. Progress in the last one year built upon our optimized pipeline for generating biomass for various functional genomics studies and utilized improved genetic methods. Numerous additional transcriptomics data sets were added to our compendium of stress response studies. These included peroxide stress, low oxygen stress, high and low pH stress and alteration of growth conditions (e.g presence of methionine, alternate electron donors etc). To understand how genotype and environment interact to determine the phenotype and fitness of an organism, a long-term evolution experiment was also conducted to examine the dynamics and adaptation of D. vulgaris under extended salt exposure. For many of these stresses iTRAQ based quantitative proteomics and CE-MS based metabolite studies were also conducted. Improved genetic methods were employed to create several critical knock out mutants (e.g. echA, qmoABC and tatA) and several were characterized via growth and transcriptomics studies. Progress was also made in extending transcriptomics analysis to examine alternate D. vulgaris physiological states such as in biofilms and growth in syntrophic co-culture with Methanococcus maripaludis. Methods to conduct iTRAQ proteomics and stable isotopomer (13C) based metabolic flux analysis were also developed for studying co-cultures. A novel FTICR-MS based method for a comparative 12C/13C based metabolite analysis is being developed and will enab

The overview of changes in protein levels or states in response to a growth condition, stress, mutation or metabolic engineering is invaluable in understanding the physiology of a microbial system. The lipid profile of the cell is similarly a valuable diagnostic of the cellular response and health, especially in context of survival in a fluctuating environment. To obtain comprehensive cellular models, post-transcriptional cell wide surveys at the levels of proteins and lipids are required. Both these fields have been greatly bolstered by the development of high throughput methods using mass spectrometry. Multiple strategies now exist for the identification of proteins, and numerous workflows to quantify protein abundance have also been developed. Cellular profiling such as these allows us to assess the potential of a microbial system for environmental applications such as bioremediation and bio-energy.

Though considered obligate anaerobes for many years after their discovery, sulfate reducing bacteria like Desulfovibrio vulgaris Hildenborough (DvH) are found in environments with very low sulfate and in many environments that are regularly exposed to oxygen or are normally aerobic. The best growth condition for DvH, measured as increase in biomass, remains a completely anaerobic environment. However, DvH is clearly able to tolerate sub-aerobic environments and can survive exposure to air for up to 20 days. Controlled experiments were conducted to expose DvH to aerobic and microaerobic conditions (0.1% O2). Cell-wide responses were monitored via transcriptomics and proteomics measurements. Microaerobic conditions caused an overall decrease in growth without affecting the viability of the bacterium. Cellular responses to microaerobic conditions were mild and primarily included up-regulation of the putative PerR regulon, but other known oxidative stress response candidates remained unchanged. Other transcripts that show an expression profile similar to the PerR regulon genes included the cydA/B operon, encoding putative oxidative phosphorylation proteins. However, comparison with data from prior DvH functional genomics studies suggested that many of these changes could be part of a general stress response in DvH. In contrast, exposure to air produced drastic changes at both the transcriptome and proteome levels and had a detrimental effect on both growth and viability of DvH. During aerobic stress, increases in proteases and chaperones signified air exposure to be a very harsh stress in DvH. However, quantitative proteomics also indicated an accumulation of superoxide-dismutase, catalase as well as ferritins and thioredoxins, and these candidates may be critical for the survival of the small fraction of cells which survive air exposure. Our results indicated that DvH has very different responses towards microaerobic vs. aerobic exposure. Growth of DvH strains under th

Though considered obligate anaerobes for many years after their discovery, sulfate reducing bacteria like Desulfovibrio vulgaris Hildenborough (DvH) are found in environments with very low sulfate and in many environments that are regularly exposed to oxygen or are normally aerobic. The best growth condition for DvH, measured as increase in biomass, remains a completely anaerobic environment. However, DvH is clearly able to tolerate sub-aerobic environments and can survive exposure to air for up to 20 days. Controlled experiments were conducted to expose DvH to aerobic and microaerobic conditions (0.1% O2). Cell-wide responses were monitored via transcriptomics and proteomics measurements. Microaerobic conditions caused an overall decrease in growth without affecting the viability of the bacterium. Cellular responses to microaerobic conditions were mild and primarily included up-regulation of the putative PerR regulon, but other known oxidative stress response candidates remained unchanged. Other transcripts that show an expression profile similar to the PerR regulon genes included the cydA/B operon, encoding putative oxidative phosphorylation proteins. However, comparison with data from prior DvH functional genomics studies suggested that many of these changes could be part of a general stress response in DvH. In contrast, exposure to air produced drastic changes at both the transcriptome and proteome levels and had a detrimental effect on both growth and viability of DvH. During aerobic stress, increases in proteases and chaperones signified air exposure to be a very harsh stress in DvH. However, quantitative proteomics also indicated an accumulation of superoxide-dismutase, catalase as well as ferritins and thioredoxins, and these candidates may be critical for the survival of the small fraction of cells which survive air exposure. Our results indicated that DvH has very different responses towards microaerobic vs. aerobic exposure. Growth of DvH strains under th

The ability of Desulfovibrio vulgaris Hildenborough to reduce, and therefore contain, toxic and radioactive metal waste has made all factors that affect the physiology of this organism of great interest. Increased salinity is an important and frequent fluctuation faced by D. vulgaris in its natural habitat. In liquid culture, exposure to excess salt resulted in striking elongation of D. vulgaris cells. Using data from transcriptomics, proteomics, metabolite assays, phospholipid fatty acid profiling, and electron microscopy, we used a systems approach to explore the effects of excess NaCl on D. vulgaris. In this study we demonstrated that import of osmoprotectants, such as glycine betaine and ectoine, is the primary mechanism used by D. vulgaris to counter hyperionic stress. Several efflux systems were also highly up-regulated, as was the ATP synthesis pathway. Increases in the levels of both RNA and DNA helicases suggested that salt stress affected the stability of nucleic acid base pairing. An overall increase in the level of branched fatty acids indicated that there were changes in cell wall fluidity. The immediate response to salt stress included up-regulation of chemotaxis genes, although flagellar biosynthesis was down-regulated. Other down-regulated systems included lactate uptake permeases and ABC transport systems. The results of an extensive NaCl stress analysis were compared with microarray data from a KCl stress analysis, and unlike many other bacteria, D. vulgaris responded similarly to the two stresses. Integration of data from multiple methods allowed us to develop a conceptual model for the salt stress response in D. vulgaris that can be compared to those in other microorganisms.

The ability of Desulfovibrio vulgaris Hildenborough to reduce, and therefore contain, toxic and radioactive metal waste has made all factors that affect the physiology of this organism of great interest. Increased salinity is an important and frequent fluctuation faced by D. vulgaris in its natural habitat. In liquid culture, exposure to excess salt resulted in striking elongation of D. vulgaris cells. Using data from transcriptomics, proteomics, metabolite assays, phospholipid fatty acid profiling, and electron microscopy, we used a systems approach to explore the effects of excess NaCl on D. vulgaris. In this study we demonstrated that import of osmoprotectants, such as glycine betaine and ectoine, is the primary mechanism used by D. vulgaris to counter hyperionic stress. Several efflux systems were also highly up-regulated, as was the ATP synthesis pathway. Increases in the levels of both RNA and DNA helicases suggested that salt stress affected the stability of nucleic acid base pairing. An overall increase in the level of branched fatty acids indicated that there were changes in cell wall fluidity. The immediate response to salt stress included up-regulation of chemotaxis genes, although flagellar biosynthesis was down-regulated. Other down-regulated systems included lactate uptake permeases and ABC transport systems. The results of an extensive NaCl stress analysis were compared with microarray data from a KCl stress analysis, and unlike many other bacteria, D. vulgaris responded similarly to the two stresses. Integration of data from multiple methods allowed us to develop a conceptual model for the salt stress response in D. vulgaris that can be compared to those in other microorganisms.