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1.
J Ind Microbiol Biotechnol ; 50(1)2023 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-37791393

RESUMO

Microbial bioproduction often faces challenges related to populational heterogeneity, where cells exhibit varying biosynthesis capabilities. Bioproduction heterogeneity can stem from genetic and non-genetic factors, resulting in decreased titer, yield, stability, and reproducibility. Consequently, understanding and controlling bioproduction heterogeneity are crucial for enhancing the economic competitiveness of large-scale biomanufacturing. In this review, we provide a comprehensive overview of current understandings of the various mechanisms underlying bioproduction heterogeneity. Additionally, we examine common strategies for controlling bioproduction heterogeneity based on these mechanisms. By implementing more robust measures to mitigate heterogeneity, we anticipate substantial enhancements in the scalability and stability of bioproduction processes. ONE-SENTENCE SUMMARY: This review summarizes current understandings of different mechanisms of bioproduction heterogeneity and common control strategies based on these mechanisms.


Assuntos
Fermentação , Reprodutibilidade dos Testes
2.
Int J Mol Sci ; 24(7)2023 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-37047388

RESUMO

High molecular weight (MW), highly repetitive protein polymers are attractive candidates to replace petroleum-derived materials as these protein-based materials (PBMs) are renewable, biodegradable, and have outstanding mechanical properties. However, their high MW and highly repetitive sequence features make them difficult to synthesize in fast-growing microbial cells in sufficient amounts for real applications. To overcome this challenge, various methods were developed to synthesize repetitive PBMs. Here, we review recent strategies in the construction of repetitive genes, expression of repetitive proteins from circular mRNAs, and synthesis of repetitive proteins by ligation and protein polymerization. We discuss the advantages and limitations of each method and highlight future directions that will lead to scalable production of highly repetitive PBMs for a wide range of applications.


Assuntos
Polímeros , Proteínas , Peso Molecular , Sequências Repetitivas de Ácido Nucleico
3.
Molecules ; 28(13)2023 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-37446650

RESUMO

Hydrogels made from proteins are attractive materials for diverse medical applications, as they are biocompatible, biodegradable, and amenable to chemical and biological modifications. Recent advances in protein engineering, synthetic biology, and material science have enabled the fine-tuning of protein sequences, hydrogel structures, and hydrogel mechanical properties, allowing for a broad range of biomedical applications using protein hydrogels. This article reviews recent progresses on protein hydrogels with special focus on those made of microbially produced proteins. We discuss different hydrogel formation strategies and their associated hydrogel properties. We also review various biomedical applications, categorized by the origin of protein sequences. Lastly, current challenges and future opportunities in engineering protein-based hydrogels are discussed. We hope this review will inspire new ideas in material innovation, leading to advanced protein hydrogels with desirable properties for a wide range of biomedical applications.


Assuntos
Materiais Biocompatíveis , Hidrogéis , Materiais Biocompatíveis/química , Hidrogéis/química , Proteínas , Engenharia Tecidual
4.
Metab Eng ; 74: 11-23, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36058465

RESUMO

Bacterial fatty acids (FAs) are an essential component of the cellular membrane and are an important source of renewable chemicals as they can be converted to fatty alcohols, esters, ketones, and alkanes, and used as biofuels, detergents, lubricants, and commodity chemicals. Most prior FA bioconversions have been performed on the carboxylic acid group. Modification of the FA hydrocarbon chain could substantially expand the structural and functional diversity of FA-derived products. Additionally, the effects of such modified FAs on the growth and metabolic state of their producing cells are not well understood. Here we engineer novel Escherichia coli phospholipid biosynthetic pathways, creating strains with distinct FA profiles enriched in ω7-unsaturated FAs (ω7-UFAs, 75%), Δ5-unsaturated FAs (Δ5-UFAs, 60%), cyclopropane FAs (CFAs, 55%), internally-branched FAs (IBFAs, 40%), and Δ5,ω7-double unsaturated FAs (DUFAs, 46%). Although bearing drastically different FA profiles in phospholipids, UFA, CFA, and IBFA enriched strains display wild-type-like phenotypic profiling and growth. Transcriptomic analysis reveals DUFA production drives increased differential expression and the induction of the fur iron starvation transcriptional cascade, but higher TCA cycle activation compared to the UFA producing strain. This likely reflects a slight cost imparted for DUFA production, which resulted in lower maximum growth in some, but not all, environmental conditions. The IBFA-enriched strain was further engineered to produce free IBFAs, releasing 96 mg/L free IBFAs from 154 mg/L of the total cellular IBFA pool. This work has resulted in significantly altered FA profiles of membrane lipids in E. coli, greatly increasing our understanding of the effects of FA structure diversity on the transcriptome, growth, and ability to react to stress.


Assuntos
Escherichia coli , Fosfolipídeos , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Ácidos Graxos/genética , Biocombustíveis , Ácidos Graxos Insaturados/genética
5.
BMC Genomics ; 22(1): 149, 2021 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-33653272

RESUMO

BACKGROUND: Cell-to-cell variation in gene expression strongly affects population behavior and is key to multiple biological processes. While codon usage is known to affect ensemble gene expression, how codon usage influences variation in gene expression between single cells is not well understood. RESULTS: Here, we used a Sort-seq based massively parallel strategy to quantify gene expression variation from a green fluorescent protein (GFP) library containing synonymous codons in Escherichia coli. We found that sequences containing codons with higher tRNA Adaptation Index (TAI) scores, and higher codon adaptation index (CAI) scores, have higher GFP variance. This trend is not observed for codons with high Normalized Translation Efficiency Index (nTE) scores nor from the free energy of folding of the mRNA secondary structure. GFP noise, or squared coefficient of variance (CV2), scales with mean protein abundance for low-abundant proteins but does not change at high mean protein abundance. CONCLUSIONS: Our results suggest that the main source of noise for high-abundance proteins is likely not originating at translation elongation. Additionally, the drastic change in mean protein abundance with small changes in protein noise seen from our library implies that codon optimization can be performed without concerning gene expression noise for biotechnology applications.


Assuntos
Biossíntese de Proteínas , RNA de Transferência , Códon/genética , Uso do Códon , Escherichia coli/genética , RNA de Transferência/genética
6.
Metab Eng ; 63: 126-140, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32927059

RESUMO

Metabolic engineering has allowed the production of a diverse number of valuable chemicals using microbial organisms. Many biological challenges for improving bio-production exist which limit performance and slow the commercialization of metabolically engineered systems. Dynamic metabolic engineering is a rapidly developing field that seeks to address these challenges through the design of genetically encoded metabolic control systems which allow cells to autonomously adjust their flux in response to their external and internal metabolic state. This review first discusses theoretical works which provide mechanistic insights and design choices for dynamic control systems including two-stage, continuous, and population behavior control strategies. Next, we summarize molecular mechanisms for various sensors and actuators which enable dynamic metabolic control in microbial systems. Finally, important applications of dynamic control to the production of several metabolite products are highlighted, including fatty acids, aromatics, and terpene compounds. Altogether, this review provides a comprehensive overview of the progress, advances, and prospects in the design of dynamic control systems for improved titer, rate, and yield metrics in metabolic engineering.


Assuntos
Engenharia Metabólica
7.
PLoS Comput Biol ; 16(1): e1007643, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32004314

RESUMO

For a genetically identical microbial population, multi-gene expression in various environments requires effective allocation of limited resources and precise control of heterogeneity among individual cells. However, it is unclear how resource allocation and cell-to-cell variation jointly shape the overall performance. Here we demonstrate a Simpson's paradox during overexpression of multiple genes: two competing proteins in single cells correlated positively for every induction condition, but the overall correlation was negative. Yet this phenomenon was not observed between two competing mRNAs in single cells. Our analytical framework shows that the phenomenon arises from competition for translational resource, with the correlation modulated by both mRNA and ribosome variability. Thus, heterogeneity plays a key role in single-cell multi-gene expression and provides the population with an evolutionary advantage, as demonstrated in this study.


Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Expressão Gênica/genética , Genes Bacterianos/genética , Biologia Computacional , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fenômenos Genéticos/genética , Modelos Estatísticos , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
8.
Int J Mol Sci ; 22(19)2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34639037

RESUMO

Amyloids are self-assembled protein aggregates that take cross-ß fibrillar morphology. Although some amyloid proteins are best known for their association with Alzheimer's and Parkinson's disease, many other amyloids are found across diverse organisms, from bacteria to humans, and they play vital functional roles. The rigidity, chemical stability, high aspect ratio, and sequence programmability of amyloid fibrils have made them attractive candidates for functional materials with applications in environmental sciences, material engineering, and translational medicines. This review focuses on recent advances in fabricating various types of macroscopic functional amyloid materials. We discuss different design strategies for the fabrication of amyloid hydrogels, high-strength materials, composite materials, responsive materials, extracellular matrix mimics, conductive materials, and catalytic materials.


Assuntos
Amiloide/química , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Aminoácidos/química , Amiloide/ultraestrutura , Proteínas Amiloidogênicas/química , Amiloidose/etiologia , Amiloidose/metabolismo , Amiloidose/patologia , Matriz Extracelular/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade
9.
Metab Eng ; 55: 120-130, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31271774

RESUMO

Rhodococcus opacus PD630 metabolizes aromatic substrates and naturally produces branched-chain lipids, which are advantageous traits for lignin valorization. To provide insights into its lignocellulose hydrolysate utilization, we performed 13C-pathway tracing, 13C-pulse-tracing, transcriptional profiling, biomass composition analysis, and metabolite profiling in conjunction with 13C-metabolic flux analysis (13C-MFA) of phenol metabolism. We found that 1) phenol is metabolized mainly through the ortho-cleavage pathway; 2) phenol utilization requires a highly active TCA cycle; 3) NADPH is generated mainly via NADPH-dependent isocitrate dehydrogenase; 4) active cataplerotic fluxes increase plasticity in the TCA cycle; and 5) gluconeogenesis occurs partially through the reversed Entner-Doudoroff pathway (EDP). We also found that phenol-fed R. opacus PD630 generally has lower sugar phosphate concentrations (e.g., fructose 1,6-bisphosphatase) compared to metabolite pools in 13C-glucose-fed Escherichia coli (set as internal standards), while its TCA metabolites (e.g., malate, succinate, and α-ketoglutarate) accumulate intracellularly with measurable succinate secretion. In addition, we found that phenol utilization was inhibited by benzoate, while catabolite repressions by other tested carbon substrates (e.g., glucose and acetate) were absent in R. opacus PD630. Three adaptively-evolved strains display very different growth rates when fed with phenol as a sole carbon source, but they maintain a conserved flux network. These findings improve our understanding of R. opacus' metabolism for future lignin valorization.


Assuntos
Proteínas de Bactérias , Evolução Molecular Direcionada , Redes e Vias Metabólicas , Fenol/metabolismo , Rhodococcus , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Rhodococcus/genética , Rhodococcus/metabolismo , Biologia de Sistemas
10.
Biomacromolecules ; 20(5): 2015-2023, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-30995840

RESUMO

Because of their association with debilitating diseases and their potential applications in developing novel bionanomaterials, highly ordered amyloid fibrils have recently received considerable attention. While many studies have thus far focused on amyloid fibrils made with short peptides containing just one steric zipper-forming segment of native amyloid proteins, the self-assembly of proteins containing multiple steric zipper-forming segments has been rarely explored. Here we develop a strategy to create four block polypeptides, each containing 16 repeats of a zipper-forming segment from four different amyloid morphological classes. All four block polypeptides self-assemble into fibrils that display the cross-ß structure characteristic of amyloids. These amyloid-spider silk block polypeptides displayed fast self-assembly kinetics, and their fibrils exhibited high thermal stability. These novel synthetic amyloids provide insights into the self-assembly of proteins containing multiple zipper-forming segments, and our approach of creating block polypeptide fibrils could be used to expand the capability of amyloid-based bionanomaterials.


Assuntos
Amiloide/química , Fibroínas/química , Zíper de Leucina , Conformação Proteica em Folha beta , Desnaturação Proteica , Multimerização Proteica
11.
Nat Chem Biol ; 12(5): 339-44, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26999780

RESUMO

Biosynthesis enables renewable production of manifold compounds, yet often biosynthetic performance must be improved for it to be economically feasible. Nongenetic, cell-to-cell variations in protein and metabolite concentrations are naturally inherent, suggesting the existence of both high- and low-performance variants in all cultures. Although having an intrinsic source of low performers might cause suboptimal ensemble biosynthesis, the existence of high performers suggests an avenue for performance enhancement. Here we develop in vivo population quality control (PopQC) to continuously select for high-performing, nongenetic variants. We apply PopQC to two biosynthetic pathways using two alternative design principles and demonstrate threefold enhanced production of both free fatty acid (FFA) and tyrosine. We confirm that PopQC improves ensemble biosynthesis by selecting for nongenetic high performers. Additionally, we use PopQC in fed-batch FFA production and achieve 21.5 g l(-1) titer and 0.5 g l(-1) h(-1) productivity. Given the ubiquity of nongenetic variation, PopQC should be applicable to a variety of metabolic pathways for enhanced biosynthesis.


Assuntos
Escherichia coli/citologia , Escherichia coli/fisiologia , Ácidos Graxos não Esterificados/biossíntese , Tirosina/biossíntese , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Engenharia Metabólica , Redes e Vias Metabólicas
12.
Biotechnol Bioeng ; 115(9): 2305-2314, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29896914

RESUMO

The oxygenic photosynthetic bacterium Synechocystis sp. PCC 6803 (S6803) is a model cyanobacterium widely used for fundamental research and biotechnology applications. Due to its polyploidy, existing methods for genome engineering of S6803 require multiple rounds of selection to modify all genome copies, which is time-consuming and inefficient. In this study, we engineered the Cas9 tool for one-step, segregation-free genome engineering. We further used our Cas9 tool to delete three of seven S6803 native plasmids. Our results show that all three small-size native plasmids, but not the large-size native plasmids, can be deleted with this tool. To further facilitate heterologous gene expression in S6803, a shuttle vector based on the native plasmid pCC5.2 was created. The shuttle vector can be introduced into Cas9-containing S6803 in one step without requiring segregation and can be stably maintained without antibiotic pressure for at least 30 days. Moreover, genes encoded on the shuttle vector remain functional after 30 days of continuous cultivation without selective pressure. Thus, this study provides a set of new tools for rapid modification of the S6803 genome and for stable expression of heterologous genes, potentially facilitating both fundamental research and biotechnology applications using S6803.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Edição de Genes/métodos , Vetores Genéticos , Genética Microbiana/métodos , Plasmídeos , Synechocystis/genética , Expressão Gênica , Instabilidade Genômica , Recombinação Genética
13.
Biomacromolecules ; 19(9): 3853-3860, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30080972

RESUMO

Despite significant efforts to engineer their heterologous production, recombinant spider silk proteins (spidroins) have yet to replicate the unparalleled combination of high strength and toughness exhibited by natural spider silks, preventing their use in numerous mechanically demanding applications. To overcome this long-standing challenge, we have developed a synthetic biology approach combining standardized DNA part assembly and split intein-mediated ligation to produce recombinant spidroins of previously unobtainable size (556 kDa), containing 192 repeat motifs of the Nephila clavipes dragline spidroin. Fibers spun from our synthetic spidroins are the first to fully replicate the mechanical performance of their natural counterparts by all common metrics, i.e., tensile strength (1.03 ± 0.11 GPa), modulus (13.7 ± 3.0 GPa), extensibility (18 ± 6%), and toughness (114 ± 51 MJ/m3). The developed process reveals a path to more dependable production of high-performance silks for mechanically demanding applications while also providing a platform to facilitate production of other high-performance natural materials.


Assuntos
Fibroínas/química , Resistência à Tração , Elasticidade , Fibroínas/genética , Fibroínas/normas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
14.
Microb Cell Fact ; 17(1): 74, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29764418

RESUMO

BACKGROUND: Despite its ability to grow and produce high-value molecules using renewable carbon sources, two main factors must be improved to use Burkholderia sacchari as a chassis for bioproduction at an industrial scale: first, the lack of molecular tools to engineer this organism and second, the inherently slow growth rate and poly-3-hydroxybutyrate [P(3HB)] production using xylose. In this work, we have addressed both factors. RESULTS: First, we adapted a set of BglBrick plasmids and showed tunable expression in B. sacchari. Finally, we assessed growth rate and P(3HB) production through overexpression of xylose transporters, catabolic or regulatory genes. Overexpression of xylR significantly improved growth rate (55.5% improvement), polymer yield (77.27% improvement), and resulted in 71% of cell dry weight as P(3HB). CONCLUSIONS: These values are unprecedented for P(3HB) accumulation using xylose as a sole carbon source and highlight the importance of precise expression control for improving utilization of hemicellulosic sugars in B. sacchari.


Assuntos
Bioengenharia/métodos , Burkholderia/química , Hidroxibutiratos/química , Poliésteres/química , Xilose/metabolismo
15.
Nature ; 488(7411): 320-8, 2012 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-22895337

RESUMO

Advanced biofuels produced by microorganisms have similar properties to petroleum-based fuels, and can 'drop in' to the existing transportation infrastructure. However, producing these biofuels in yields high enough to be useful requires the engineering of the microorganism's metabolism. Such engineering is not based on just one specific feedstock or host organism. Data-driven and synthetic-biology approaches can be used to optimize both the host and pathways to maximize fuel production. Despite some success, challenges still need to be met to move advanced biofuels towards commercialization, and to compete with more conventional fuels.


Assuntos
Biocombustíveis/provisão & distribuição , Engenharia Genética , Microbiologia , Álcoois/química , Álcoois/metabolismo , Biocombustíveis/economia , Biomassa , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Petróleo/metabolismo , Petróleo/estatística & dados numéricos , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Biologia Sintética , Terpenos/química , Terpenos/metabolismo , Meios de Transporte
16.
J Ind Microbiol Biotechnol ; 45(7): 535-543, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29380150

RESUMO

Advances in metabolic engineering have led to the synthesis of a wide variety of valuable chemicals in microorganisms. The key to commercializing these processes is the improvement of titer, productivity, yield, and robustness. Traditional approaches to enhancing production use the "push-pull-block" strategy that modulates enzyme expression under static control. However, strains are often optimized for specific laboratory set-up and are sensitive to environmental fluctuations. Exposure to sub-optimal growth conditions during large-scale fermentation often reduces their production capacity. Moreover, static control of engineered pathways may imbalance cofactors or cause the accumulation of toxic intermediates, which imposes burden on the host and results in decreased production. To overcome these problems, the last decade has witnessed the emergence of a new technology that uses synthetic regulation to control heterologous pathways dynamically, in ways akin to regulatory networks found in nature. Here, we review natural metabolic control strategies and recent developments in how they inspire the engineering of dynamically regulated pathways. We further discuss the challenges of designing and engineering dynamic control and highlight how model-based design can provide a powerful formalism to engineer dynamic control circuits, which together with the tools of synthetic biology, can work to enhance microbial production.


Assuntos
Proteínas de Bactérias/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , Biologia Sintética/métodos , Técnicas Biossensoriais , Fermentação
17.
Biotechnol Bioeng ; 114(2): 463-467, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27497084

RESUMO

Our recent 13 C-metabolic flux analysis (13 C-MFA) study indicates that energy metabolism becomes a rate-limiting factor for fatty acid overproduction in E. coli strains (after "Push-Pull-Block" based genetic modifications). To resolve this bottleneck, Vitreoscilla hemoglobin (VHb, a membrane protein facilitating O2 transport) was introduced into a fatty-acid-producing strain to promote oxygen supply and energy metabolism. The resulting strain, FAV50, achieved 70% percent higher fatty acid titer than the parent strain in micro-aerobic shake tube cultures. In high cell-density bioreactor fermentations, FAV50 achieved free fatty acids at a titer of 7.02 g/L (51% of the theoretical yield). In addition to "Push-Pull-Block-Power" strategies, our experiments and flux balance analysis also revealed the fatty acid over-producing strain is sensitive to metabolic burden and oxygen influx, and thus a careful evaluation of the cost-benefit tradeoff with the guidance of fluxome analysis will be fundamental for the rational design of synthetic biology strains. Biotechnol. Bioeng. 2017;114: 463-467. © 2016 Wiley Periodicals, Inc.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Ácidos Graxos/metabolismo , Engenharia Metabólica/métodos , Oxigênio/metabolismo , Proteínas Recombinantes/genética , Hemoglobinas Truncadas/genética , Proteínas de Bactérias/metabolismo , Reatores Biológicos/microbiologia , Metabolismo Energético , Escherichia coli/metabolismo , Ácidos Graxos/análise , Fermentação , Análise do Fluxo Metabólico , Proteínas Recombinantes/metabolismo , Biologia Sintética , Hemoglobinas Truncadas/metabolismo
18.
Metab Eng ; 38: 148-158, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27421620

RESUMO

Branched-chain fatty acids (BCFAs) are key precursors of branched-chain fuels, which have cold-flow properties superior to straight chain fuels. BCFA production in Gram-negative bacterial hosts is inherently challenging because it competes directly with essential and efficient straight-chain fatty acid (SCFA) biosynthesis. Previously, Escherichia coli strains engineered for BCFA production also co-produced a large percentage of SCFA, complicating efficient isolation of BCFA. Here, we identified a key bottleneck in BCFA production: incomplete lipoylation of 2-oxoacid dehydrogenases. We engineered two protein lipoylation pathways that not only restored 2-oxoacid dehydrogenase lipoylation, but also increased BCFA production dramatically. E. coli expressing an optimized lipoylation pathway produced 276mg/L BCFA, comprising 85% of the total free fatty acids (FFAs). Furthermore, we fine-tuned BCFA branch positions, yielding strains specifically producing ante-iso or odd-chain iso BCFA as 77% of total FFA, separately. When coupled with an engineered branched-chain amino acid pathway to enrich the branched-chain α-ketoacid pool, BCFA can be produced from glucose at 181mg/L and 72% of total FFA. While E. coli can metabolize BCFAs, we demonstrated that they are not incorporated into the cell membrane, allowing our system to produce a high percentage of BCFA without affecting membrane fluidity. Overall, this work establishes a platform for high percentage BCFA production, providing the basis for efficient and specific production of a variety of branched-chain hydrocarbons in engineered bacterial hosts.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Ácidos Graxos/biossíntese , Glucose/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/fisiologia , Oxirredutases/genética , Vias Biossintéticas/fisiologia , Proteínas de Escherichia coli/genética , Ácidos Graxos/genética , Melhoramento Genético/métodos , Lipogênese/fisiologia , Oxirredutases/metabolismo
19.
Environ Sci Technol ; 50(5): 2514-21, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26824511

RESUMO

Graphene oxide (GO) materials have demonstrated considerable potential in next-generation water treatment membrane-based technologies, which include antimicrobial applications. GO antimicrobial properties can be further enhanced by preloading or chemically generating surface-associated nanoscale silver particles (nAg). However, for these systems, enhanced antimicrobial functionality decreases over time as a function of Ag mass loss via dissolution (as Ag(+)). In this work, we demonstrate facile photocatalytic in situ synthesis of nAg particles by crumpled GO-TiO2 (GOTI) nanocomposites as an approach to (re)generate, and thus maintain, enhanced antimicrobial activity over extended operation times. The described photocatalytic formation process is highly efficient and relatively fast, producing nAg particles over a size range of 40 to 120 nm and with active (111) planes. Additionally, we show in situ surface-based photocatalyzed synthesis of nAg particles at the surface of GOTI nanocomposite membrane assemblies, allowing for simultaneous filtration and disinfection. With ca. 3 log inactivation for both Escherichia coli and Bacillus subtilis, the described membrane assemblies with in situ formed nAg demonstrate enhanced antimicrobial activity compared to the GOTI membrane surface or the support membrane alone. Under typical conditions, the working and operational time (Ag dissolution time) is calculated to be over 2 orders of magnitude higher than the loading (synthesis) time (e.g., 123 h versus 0.5 h, respectively). Taken together, results highlight the described material-based process as a potentially novel antifouling membrane technology.


Assuntos
Filtração/instrumentação , Grafite/química , Nanopartículas/química , Fotoquímica/métodos , Prata/química , Anti-Infecciosos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Desinfecção/métodos , Escherichia coli/efeitos dos fármacos , Membranas Artificiais , Nanocompostos/química , Titânio/química , Purificação da Água/métodos
20.
Metab Eng ; 31: 35-43, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26142692

RESUMO

Quantification and regulation of pathway metabolites is crucial for optimization of microbial production bioprocesses. Genetically encoded biosensors provide the means to couple metabolite sensing to several outputs invaluable for metabolic engineering. These include semi-quantification of metabolite concentrations to screen or select strains with desirable metabolite characteristics, and construction of dynamic metabolite-regulated pathways to enhance production. Taking inspiration from naturally occurring systems, biosensor functions are based on highly diverse mechanisms including metabolite responsive transcription factors, two component systems, cellular stress responses, regulatory RNAs, and protein activities. We review recent developments in biosensors in each of these mechanistic classes, with considerations towards how these sensors are engineered, how new sensing mechanisms have led to improved function, and the advantages and disadvantages of each of these sensing mechanisms in relevant applications. We particularly highlight recent examples directly using biosensors to improve microbial production, and the great potential for biosensors to further inform metabolic engineering practices.


Assuntos
Técnicas Biossensoriais , Engenharia Metabólica/métodos , Animais , Humanos , Proteínas/metabolismo , RNA/metabolismo , Estresse Fisiológico , Biologia Sintética , Fatores de Transcrição/metabolismo
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