RESUMEN
Natural and pure p-coumaric acid has valuable applications, and it can be produced via bioprocessing. However, fermentation processes have so far been unable to provide sufficient production metrics, while a biocatalytic process decoupling growth and production historically showed much promise. This biocatalytic process is revisited in order to tackle product inhibition of the key enzyme tyrosine ammonia lyase. In situ product removal is proposed as a possible solution, and a polymer/salt aqueous two-phase system is identified as a suitable system for extraction of p-coumaric acid from an alkaline solution, with a partition coefficient of up to 13. However, a 10 % salt solution was found to reduce tyrosine ammonia lyase activity by 19 %, leading to the need for a more dilute system. The cloud points of two aqueous two-phase systems at 40 °C and pHâ 10 were found to be 3.8 % salt and 9.5 % polymer, and a 5 % potassium phosphate and 12.5 % poly(ethylene glycol-ran-propylene glycol) mW~2500 system was selected for inâ situ product removal. An immobilized tyrosine ammonia lyase biocatalyst in this aqueous two-phase system produced up to 33â g/L p-coumaric acid within 24â hours, a 1.9-fold improvement compared to biocatalysis without inâ situ product removal.
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Bio-based production of fuels, chemicals and materials is needed to replace current fossil fuel based production. However, bio-based production processes are very costly, so the process needs to be as efficient as possible. Developments in synthetic biology tools has made it possible to dynamically modulate cellular metabolism during a fermentation. This can be used towards two-stage fermentations, where the process is separated into a growth and a production phase, leading to more efficient feedstock utilization and thus potentially lower costs. This article reviews the current status and some recent results in application of synthetic biology tools towards two-stage fermentations, and compares this approach to pre-existing ones, such as nutrient limitation and addition of toxins/inhibitors.
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Parageobacillus thermoglucosidasius represents a thermophilic, facultative anaerobic bacterial chassis, with several desirable traits for metabolic engineering and industrial production. To further optimize strain productivity, a systems level understanding of its metabolism is needed, which can be facilitated by a genome-scale metabolic model. Here, we present p-thermo, the most complete, curated and validated genome-scale model (to date) of Parageobacillus thermoglucosidasius NCIMB 11955. It spans a total of 890 metabolites, 1175 reactions and 917 metabolic genes, forming an extensive knowledge base for P. thermoglucosidasius NCIMB 11955 metabolism. The model accurately predicts aerobic utilization of 22 carbon sources, and the predictive quality of internal fluxes was validated with previously published 13C-fluxomics data. In an application case, p-thermo was used to facilitate more in-depth analysis of reported metabolic engineering efforts, giving additional insight into fermentative metabolism. Finally, p-thermo was used to resolve a previously uncharacterised bottleneck in anaerobic metabolism, by identifying the minimal required supplemented nutrients (thiamin, biotin and iron(III)) needed to sustain anaerobic growth. This highlights the usefulness of p-thermo for guiding the generation of experimental hypotheses and for facilitating data-driven metabolic engineering, expanding the use of P. thermoglucosidasius as a high yield production platform.
Asunto(s)
Bacillaceae , Compuestos Férricos , Anaerobiosis , Ingeniería MetabólicaRESUMEN
Inducible expression systems can be applied to control the expression of proteins or biochemical pathways in cell factories. However, several of the established systems require the addition of expensive inducers, making them unfeasible for large-scale production. Here, we establish a genome integrated trp-T7 expression system where tryptophan can be used to control the induction of a gene or a metabolic pathway. We show that the initiation of gene expression from low- and high-copy vectors can be tuned by varying the initial concentration of tryptophan or yeast extract, and that expression is tightly regulated and homogenous when compared with the commonly used lac-T7 system. Finally, we apply the trp-T7 expression system for the production of l-serine, where we reach titers of 26 g/L in fed-batch fermentation.
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Escherichia coli , Ingeniería Metabólica/métodos , Regiones Promotoras Genéticas/genética , Reactores Biológicos/microbiología , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentación , Serina/metabolismo , Triptófano/metabolismoRESUMEN
Growth decoupling can be used to optimize the production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference to increase the production of a single-domain antibody (sdAb) by inhibiting growth during production. First, we screened 21 sgRNA targets in the purine and pyrimidine biosynthesis pathways and found that the repression of 11 pathway genes led to the increased green fluorescent protein production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve the production of two versions of an expression-optimized sdAb. Proteomics analysis of the sdAb-producing pyrF, pyrG, and cmk growth decoupling strains showed significantly decreased RpoS levels and an increase of ribosome-associated proteins, indicating that the growth decoupling strains do not enter stationary phase and maintain their capacity for protein synthesis upon growth inhibition. Finally, sdAb production was scaled up to shake-flask fermentation where the product yield was improved 2.6-fold compared to the control strain with no sgRNA target sequence. An sdAb content of 14.6% was reached in the best-performing pyrG growth decoupling strain.
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Sistemas CRISPR-Cas , Escherichia coli , Ingeniería Metabólica , Nucleótidos , Anticuerpos de Dominio Único/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Nucleótidos/biosíntesis , Nucleótidos/genética , Anticuerpos de Dominio Único/genéticaRESUMEN
BACKGROUND: Sucrose is an attractive industrial carbon source due to its abundance and the fact that it can be cheaply generated from sources such as sugarcane. However, only a few characterized Escherichia coli strains are able to metabolize sucrose, and those that can are typically slow growing or pathogenic strains. METHODS: To generate a platform strain capable of efficiently utilizing sucrose with a high growth rate, adaptive laboratory evolution (ALE) was utilized to evolve engineered E. coli K-12 MG1655 strains containing the sucrose utilizing csc genes (cscB, cscK, cscA) alongside the native sucrose consuming E. coli W. RESULTS: Evolved K-12 clones displayed an increase in growth and sucrose uptake rates of 1.72- and 1.40-fold on sugarcane juice as compared to the original engineered strains, respectively, while E. coli W clones showed a 1.4-fold increase in sucrose uptake rate without a significant increase in growth rate. Whole genome sequencing of evolved clones and populations revealed that two genetic regions were frequently mutated in the K-12 strains; the global transcription regulatory genes rpoB and rpoC, and the metabolic region related to a pyrimidine biosynthetic deficiency in K-12 attributed to pyrE expression. These two mutated regions have been characterized to confer a similar benefit when glucose is the main carbon source, and reverse engineering revealed the same causal advantages on M9 sucrose. Additionally, the most prevalent mutation found in the evolved E. coli W lineages was the inactivation of the cscR gene, the transcriptional repression of sucrose uptake genes. CONCLUSION: The generated K-12 and W platform strains, and the specific sets of mutations that enable their phenotypes, are available as valuable tools for sucrose-based industrial bioproduction in the facile E. coli chassis.
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Escherichia coli/genética , Escherichia coli/metabolismo , Sacarosa/metabolismo , Evolución Molecular Dirigida , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ingeniería Genética , Genoma Bacteriano , Glucosa/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismoRESUMEN
Given the commercial importance of the compounds produced by genetically modified organisms, there is a need for screening methods which facilitate the evaluation of newly developed strains, especially during the phase of proof-of-concept development. We report a time-efficient analysis method for the screening of bacterial strains, which enables the detection of two structurally similar secondary bacterial metabolites. By combining liquid-liquid extraction and surface-enhanced Raman scattering we were able to quantify p-coumaric acid and cinnamic acid, produced by genetically modified E. coli from tyrosine and phenylalanine, respectively. With the simple sample pre-treatment method, and by applying a partial least squares data analysis method, we simultaneously detected the analytes from four E. coli strains cultured in the presence or absence of tyrosine and phenylalanine.
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Cinamatos/análisis , Escherichia coli/clasificación , Escherichia coli/metabolismo , Extracción Líquido-Líquido/métodos , Propionatos/análisis , Espectrometría Raman/métodos , Amoníaco-Liasas/metabolismo , Ácidos Cumáricos , Escherichia coli/enzimología , Fenilalanina/metabolismo , Fenilanina Amoníaco-Liasa/metabolismo , Tirosina/metabolismoRESUMEN
Interference with genes is the foundation of reverse genetics and is key to manipulation of living cells for biomedical and biotechnological applications. However, classical genetic knockout and transcriptional knockdown technologies have different drawbacks and offer no control over existing protein levels. Here, we describe an efficient genome editing approach that affects specific protein abundances by changing the rates of both RNA synthesis and protein degradation, based on the two cross-kingdom control mechanisms CRISPRi and the N-end rule for protein stability. In addition, our approach demonstrates that CRISPRi efficiency is dependent on endogenous gene expression levels. The method has broad applications in e.g. study of essential genes and antibiotics discovery.
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Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN Bacteriano/genética , Escherichia coli/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes , Proteolisis , ARN Guía de Kinetoplastida/genéticaRESUMEN
The number of newly developed genetic variants of microbial cell factories for production of biochemicals has been rapidly growing in recent years, leading to an increased need for new screening techniques. We developed a method based on surface-enhanced Raman scattering (SERS) coupled with liquid-liquid extraction (LLE) for quantification of p-coumaric acid (pHCA) in the supernatant of genetically engineered Escherichia coli (E. coli) cultures. pHCA was measured in a dynamic range from 1 µM up to 50 µM on highly uniform SERS substrates based on leaning gold-capped nanopillars, which showed an in-wafer signal variation of only 11.7%. LLE using dichloromethane as organic phase was combined with the detection in order to increase selectivity and sensitivity by decreasing the effect of interfering compounds from the analytes of interest. The difference in pHCA production yield between three genetically engineered E. coli strains was successfully evaluated using SERS and confirmed with high-performance liquid chromatography. As this novel approach has potential to be automated and parallelized, it can be considered for high-throughput screening in metabolic engineering.
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Escherichia coli/metabolismo , Propionatos/análisis , Ácidos Cumáricos , Escherichia coli/química , Oro/química , Nanopartículas del Metal/química , Estructura Molecular , Propionatos/metabolismo , Espectrometría Raman , Propiedades de SuperficieRESUMEN
During the last few decades, great advances have been reached in high-throughput design and building of genetically engineered microbial strains, leading to a need for fast and reliable screening methods. We developed and optimized a microfluidic supported liquid membrane (SLM) extraction device and combined it with surface enhanced Raman scattering (SERS) sensing for the screening of a biological process, namely for the quantification of a bacterial secondary metabolite, p-coumaric acid (pHCA), produced by Escherichia coli. The microfluidic device proved to be robust and reusable, enabling efficient removal of interfering compounds from the real samples, reaching more than 13-fold up-concentration of the donor at 10 µL min-1 flow rate. With this method, we quantified pHCA directly from the bacterial supernatant, distinguishing between various culture conditions based on the pHCA production yield. The obtained data showed good correlation with HPLC analysis.
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Escherichia coli/química , Dispositivos Laboratorio en un Chip , Espectrometría Raman , Cromatografía Líquida de Alta Presión , Metabolismo SecundarioRESUMEN
Production of proteins and biochemicals in microbial cell factories is often limited by carbon and energy spent on excess biomass formation. To address this issue, we developed several genetic growth switches based on CRISPR interference technology. We demonstrate that growth of Escherichia coli can be controlled by repressing the DNA replication machinery, by targeting dnaA and oriC, or by blocking nucleotide synthesis through pyrF or thyA. This way, total GFP-protein production could be increased by up to 2.2-fold. Single-cell dynamic tracking in microfluidic systems was used to confirm functionality of the growth switches. Decoupling of growth from production of biochemicals was demonstrated for mevalonate, a precursor for isoprenoid compounds. Mass yield of mevalonate was increased by 41%, and production was maintained for more than 45h after activation of the pyrF-based growth switch. The developed methods represent a promising approach for increasing production yield and titer for proteins and biochemicals.
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Proteínas Bacterianas/biosíntesis , Carbono/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genes de Cambio/genética , Ingeniería Metabólica/métodos , Synechococcus/genética , Proteínas Bacterianas/genética , Redes y Vías Metabólicas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Synechococcus/metabolismo , Activación Transcripcional/genéticaRESUMEN
L-serine is a widely used amino acid that has been proposed as a potential building block biochemical. The high theoretical yield from glucose makes a fermentation based production attractive. In order to achieve this goal, serine degradation to pyruvate and glycine in E. coli MG1655 was prevented by deletion of three L-serine deaminases sdaA, sdaB, and tdcG, as well as serine hydroxyl methyl transferase (SHMT) encoded by glyA. Upon overexpression of the serine production pathway, consisting of a feedback resistant version of serA along with serB and serC, this quadruple deletion strain showed a very high serine production yield (0.45 g/g glucose) during small-scale batch fermentation in minimal medium. Serine, however, was found to be highly toxic even at low concentrations to this strain, which lead to slow growth and production during fed batch fermentation, resulting in a serine production of 8.3 g/L. The production strain was therefore evolved by random mutagenesis to achieve increased tolerance towards serine. Additionally, overexpression of eamA, a cysteine/homoserine transporter was demonstrated to increase serine tolerance from 1.6 g/L to 25 g/L. During fed batch fermentation, the resulting strain lead to the serine production titer of 11.7 g/L with yield of 0.43 g/g glucose, which is the highest yield reported so far for any organism.
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Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Metabólica/métodos , Serina/metabolismo , Transporte Biológico , Tolerancia a Medicamentos , Escherichia coli/crecimiento & desarrollo , Eliminación de Gen , Expresión Génica , Redes y Vías Metabólicas/genética , Mutación , Serina/toxicidadRESUMEN
BACKGROUND: Bacterial small RNAs (sRNAs) are recognized as posttranscriptional regulators involved in the control of bacterial lifestyle and adaptation to stressful conditions. Although chemical stress due to the toxicity of precursor and product compounds is frequently encountered in microbial bioprocessing applications, the involvement of sRNAs in this process is not well understood. We have used RNA sequencing to map sRNA expression in E. coli under chemical stress and high cell density fermentation conditions with the aim of identifying sRNAs involved in the transcriptional response and those with potential roles in stress tolerance. RESULTS: RNA sequencing libraries were prepared from RNA isolated from E. coli K-12 MG1655 cells grown under high cell density fermentation conditions or subjected to chemical stress with twelve compounds including four organic solvent-like compounds, four organic acids, two amino acids, geraniol and decanoic acid. We have discovered 253 novel intergenic transcripts with this approach, adding to the roughly 200 intergenic sRNAs previously reported in E. coli. There are eighty-four differentially expressed sRNAs during fermentation, of which the majority are novel, supporting possible regulatory roles for these transcripts in adaptation during different fermentation stages. There are a total of 139 differentially expressed sRNAs under chemical stress conditions, where twenty-nine exhibit significant expression changes in multiple tested conditions, suggesting that they may be involved in a more general chemical stress response. Among those with known functions are sRNAs involved in regulation of outer membrane proteins, iron availability, maintaining envelope homeostasis, as well as sRNAs incorporated into complex networks controlling motility and biofilm formation. CONCLUSIONS: This study has used deep sequencing to reveal a wealth of hitherto undescribed sRNAs in E. coli and provides an atlas of sRNA expression during seventeen different growth and stress conditions. Although the number of novel sRNAs with regulatory functions is unknown, several exhibit specific expression patterns during high cell density fermentation and are differentially expressed in the presence of multiple chemicals, suggesting they may play regulatory roles during these stress conditions. These novel sRNAs, together with specific known sRNAs, are candidates for improving stress tolerance and our understanding of the E. coli regulatory network during fed-batch fermentation.
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Proteínas de Escherichia coli/genética , Escherichia coli/genética , ARN Pequeño no Traducido/genética , Solventes/farmacología , Técnicas de Cultivo Celular por Lotes , Escherichia coli/metabolismo , Fermentación , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Bacteriano/genética , Análisis de Secuencia de ARN/métodosRESUMEN
Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence. We performed a comparative in vivo study, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p-coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus, a bacterial-type enzyme from the amoeba Dictyostelium discoideum, and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 µM cinnamic acid per unit of optical density at 600 nm [OD600]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p-coumaric acid in Escherichia coli (producing 440 µM p-coumaric acid OD600 unit(-1) in the medium) and in Lactococcus lactis. The enzymes were also efficient in Saccharomyces cerevisiae, where p-coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases.
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Amoníaco-Liasas/metabolismo , Bacterias/enzimología , Bacterias/metabolismo , Hidrocarburos Aromáticos/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Amoníaco-Liasas/genética , Bacterias/genética , Expresión Génica , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: One of the bottlenecks in production of biochemicals and pharmaceuticals in Saccharomyces cerevisiae is stable and homogeneous expression of pathway genes. Integration of genes into the genome of the production organism is often a preferred option when compared to expression from episomal vectors. Existing approaches for achieving stable simultaneous genome integrations of multiple DNA fragments often result in relatively low integration efficiencies and furthermore rely on the use of selection markers. RESULTS: Here, we have developed a novel method, CrEdit (CRISPR/Cas9 mediated genome Editing), which utilizes targeted double strand breaks caused by CRISPR/Cas9 to significantly increase the efficiency of homologous integration in order to edit and manipulate genomic DNA. Using CrEdit, the efficiency and locus specificity of targeted genome integrations reach close to 100% for single gene integration using short homology arms down to 60 base pairs both with and without selection. This enables direct and cost efficient inclusion of homology arms in PCR primers. As a proof of concept, a non-native ß-carotene pathway was reconstructed in S. cerevisiae by simultaneous integration of three pathway genes into individual intergenic genomic sites. Using longer homology arms, we demonstrate highly efficient and locus-specific genome integration even without selection with up to 84% correct clones for simultaneous integration of three gene expression cassettes. CONCLUSIONS: The CrEdit approach enables fast and cost effective genome integration for engineering of S. cerevisiae. Since the choice of the targeting sites is flexible, CrEdit is a powerful tool for diverse genome engineering applications.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/genética , Expresión Génica , Vectores Genéticos , Saccharomyces cerevisiae/metabolismoRESUMEN
Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site-specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome-editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user-friendly bioinformatics tool, named "CRISPy" for rapid identification of sgRNA target sequences in the CHO-K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27,553 genes and lists the number of off-target sites in the genome. In conclusion, the proven functionality of Cas9 to edit CHO genomes combined with our CRISPy database have the potential to accelerate genome editing and synthetic biology efforts in CHO cells.
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Células CHO/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Fucosiltransferasas/genética , Técnicas de Inactivación de Genes/métodos , Chaperonas Moleculares/genética , Edición de ARN , Animales , Secuencia de Bases , Cricetinae , Cricetulus , Endonucleasas/genética , Endonucleasas/metabolismo , Genoma , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación INDEL , Internet , Datos de Secuencia Molecular , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismoRESUMEN
Acetogenic gas fermentation is increasingly studied as a promising technology to upcycle carbon-rich waste gasses. Currently the product range is limited, and production yields, rates and titres for a number of interesting products do not allow for economically viable processes. By pairing process modelling and host-agnostic metabolic modelling, we compare fermentation conditions and various products to optimise the processes. The models were then used in a simulation of an industrial-scale bubble column reactor. We find that increased temperatures favour gas transfer rates, particularly for the valuable and limiting H2 , while furthermore predicting an optimal feed composition of 9:1 mol H2 to mol CO2 . Metabolically, the increased non-growth associated maintenance requirements of thermophiles favours the formation of catabolic products. To assess the expansion of the product portfolio beyond acetate, both a product volatility analysis and a metabolic pathway model were implemented. In-situ recovery of volatile products is shown to be within range for acetone but challenging due to the extensive evaporation of water, while the direct production of more valuable compounds by acetogens is metabolically unfavourable compared to acetate and ethanol. We discuss alternative approaches to overcome these challenges to utilise acetogenic CO2 fixation to produce a wider range of carbon negative chemicals.
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Dióxido de Carbono , Gases , Dióxido de Carbono/metabolismo , Fermentación , Gases/metabolismo , Acetatos/metabolismo , CarbonoRESUMEN
p-Coumaric acid (pCA) can be produced via bioprocessing and is a promising chemical precursor to making organic thin film transistors. However, the required tyrosine ammonia lyase (TAL) enzyme generally has a low specific activity and suffers from competitive product inhibition. Here we characterized the purified TAL variants from Flavobacterium johnsoniae and Herpetosiphon aurantiacus in terms of their susceptibility to product inhibition and their activity and stability across pH and temperature via initial rate experiments. FjTAL was found to be more active than previously described and to have a relatively weak affinity for pCA, but modeling revealed that product inhibition would still be problematic at industrially relevant product concentrations, due to the low solubility of the substrate tyrosine. The activity of both variants increased with temperature when tested up to 45°C, but HaTAL1 was more stable at elevated temperature. FjTAL is a promising biocatalyst for pCA production, but enzyme or bioprocess engineering are required to stabilize FjTAL and reduce product inhibition.
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Amoníaco-Liasas , Flavobacterium , Flavobacterium/genética , Amoníaco-Liasas/genética , Amoníaco-Liasas/química , TirosinaRESUMEN
The expression of correctly folded and functional heterologous proteins is important in many biotechnological production processes, whether it is enzymes, biopharmaceuticals or biosynthetic pathways for production of sustainable chemicals. For industrial applications, bacterial platform organisms, such as E. coli, are still broadly used due to the availability of tools and proven suitability at industrial scale. However, expression of heterologous proteins in these organisms can result in protein aggregation and low amounts of functional protein. This review provides an overview of the cellular mechanisms that can influence protein folding and expression, such as co-translational folding and assembly, chaperone binding, as well as protein quality control, across different model organisms. The knowledge of these mechanisms is then linked to different experimental methods that have been applied in order to improve functional heterologous protein folding, such as codon optimization, fusion tagging, chaperone co-production, as well as strain and protein engineering strategies.
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Escherichia coli , Pliegue de Proteína , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería de Proteínas , Biotecnología , Codón , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Parageobacillus thermoglucosidasius is a thermophilic Gram-positive bacterium, which is a promising host organism for sustainable bio-based production processes. However, to take full advantage of the potential of P. thermoglucosidasius, more efficient tools for genetic engineering are required. The present study describes an improved shuttle vector, which speeds up recombination-based genomic modification by incorporating a thermostable sfGFP variant into the vector backbone. This additional selection marker allows for easier identification of recombinants, thereby removing the need for several culturing steps. The novel GFP-based shuttle is therefore capable of facilitating faster metabolic engineering of P. thermoglucosidasius through genomic deletion, integration, or exchange. To demonstrate the efficiency of the new system, the GFP-based vector was utilised for deletion of the spo0A gene in P. thermoglucosidasius DSM2542. This gene is known to be a key regulator of sporulation in Bacillus subtilis, and it was therefore hypothesised that the deletion of spo0A in P. thermoglucosiadius would produce an analogous sporulation-inhibited phenotype. Subsequent analyses of cell morphology and culture heat resistance suggests that the P. thermoglucosidasius ∆spo0A strain is sporulation-deficient. This strain may be an excellent starting point for future cell factory engineering of P. thermoglucosidasius, as the formation of endospores is normally not a desired trait in large-scale production.